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After an hour-long telephone call, Putin said in a statement that Moscow and Washington were still far apart on the situation in the former Soviet republic, where he said the new authorities had taken "absolutely illegitimate decisions on the eastern, southeastern and Crimea regions.
"Russia cannot ignore calls for help and it acts accordingly, in full compliance with international law," Putin said.
Ukraine's border guards said Moscow had poured troops into the southern peninsula where Russian forces have seized control.
Serhiy Astakhov, an aide to the border guards' commander, said there were now 30,000 Russian soldiers in Crimea, compared to the 11,000 permanently based with the Russian Black Sea fleet in the port of Sevastopol before the crisis.
Putin denies that the forces with no national insignia that are surrounding Ukrainian troops in their bases are under Moscow's command, although their vehicles have Russian military plates. The West has ridiculed his assertion.
The most serious east-west confrontation since the end of the Cold War - resulting from the overthrow last month of President Viktor Yanukovich after violent protests in Kiev - escalated on Thursday (local time) when Crimea's parliament, dominated by ethnic Russians, voted to join Russia. The region's government set a referendum for March 16 - in just nine days' time.
European Union leaders and Obama denounced the referendum as illegitimate, saying it would violate Ukraine's constitution.
The head of Russia's upper house of parliament said after meeting visiting Crimean lawmakers on Friday that Crimea had a right to self-determination, and ruled out any risk of war between "the two brotherly nations".
Obama announced the first sanctions against Russia on Thursday since the start of the crisis, ordering visa bans and asset freezes against so far unidentified people deemed responsible for threatening Ukraine's sovereignty.
Japan endorsed the Western position that the actions of Russia, whose forces have seized control of the Crimean peninsula, constitute "a threat to international peace and security", after Obama spoke to Prime Minister Shinzo Abe.
China, often a Russian ally in blocking Western moves in the U.N. Security Council, was more cautious, saying that economic sanctions were not the best way to solve the crisis and avoiding comment on the legality of a Crimean referendum on secession.
The EU, Russia's biggest economic partner and energy customer, adopted a three-stage plan to try to force a negotiated solution but stopped short of immediate sanctions.
The Russian Foreign Ministry responded angrily on Friday, calling the EU decision to freeze talks on visa-free travel and on a broad new pact governing Russia-EU ties "extremely unconstructive". It warned that Moscow would retaliate against any sanctions.
Senior Ukrainian opposition politician Yulia Tymoshenko, freed from prison after Yanukovich's ouster, met German Chancellor Angela Merkel in Dublin and appealed for immediate EU sanctions against Russia, warning that Crimea might otherwise slide into a guerrilla war.
Brussels and Washington rushed to strengthen the new authorities in economically shattered Ukraine, announcing both political and financial assistance. The regional director of the International Monetary Fund said talks with Kiev on a loan agreement were going well and praised the new government's openness to economic reform and transparency.
The European Commission has said Ukraine could receive up to €11 billion ($US15 billion) in the next couple of years provided it reaches agreement with the IMF, which requires painful economic reforms like ending gas subsidies.
Promises of billions of dollars in Western aid for the Kiev government, and the perception that Russian troops are not likely to go beyond Crimea into other parts of Ukraine, have helped reverse a rout in the local hryvnia currency.
In the past two days it has traded above 9.0 to the dollar for the first time since the Crimea crisis began last week. Local dealers said emergency currency restrictions imposed last week were also supporting the hryvnia.
Russian gas monopoly Gazprom said Ukraine had not paid its $US440 million gas bill for February, bringing its arrears to $US1.89 billion and hinted it could turn off the taps as it did in 2009, when a halt in Russian deliveries to Ukraine reduced supplies to Europe during a cold snap.
In Moscow, a huge crowd gathered near the Kremlin at a government-sanctioned rally and concert billed as being "in support of the Crimean people".
Pop stars took to the stage and demonstrators held signs with slogans such as "Crimea is Russian land", "We don't trade our people for money" and "We believe in Putin".
Ukrainian Prime Minister Arseny Yatseniuk said no one in the civilised world would recognise the result of the "so-called referendum" in Crimea.
He repeated Kiev's willingness to negotiate with Russia if Moscow pulls its additional troops out of Crimea and said he had requested a telephone call with Russian Prime Minister Dmitry Medvedev.
But German Foreign Minister Frank-Walter Steinmeier said there was no clear signal that Russia was willing to join an international "contact group" with Ukraine proposed by the West to negotiate a solution to the crisis.
Despite Putin's tough words, demonstrators who have remained encamped in Kiev's central Independence Square to defend the revolution that ousted Yanukovich said they did not believe Crimea would be allowed to secede.
"We are optimists. Crimea will stand with us and we will fight for it," said Taras Yurkiv, 35, from the eastern city of Lviv. "How we will fight depends on the decisions of our leadership. If necessary, we will go with force. If you want peace, you must prepare for war."
Alexander Zaporozhets, 40, from central Ukraine's Kirovograd region, put his faith in international pressure.
"I don't think the Russians will be allowed to take Crimea from us: you can't behave like that to an independent state. We have the support of the whole world. But I think we are losing time. While the Russians are preparing, we are just talking."
Unarmed military observers from the pan-European Organisation for Security and Cooperation in Europe were blocked from entering Crimea for a second day in a row on Friday, the OSCE said on Twitter.
A U.N. special envoy who travelled to the regional capital Simferopol was surrounded by pro-Russian protesters and forced to leave on Wednesday. The United Nations said it had sent its assistant secretary-general for human rights, Ivan Simonovic, to Kiev to conduct a preliminary humans rights assessment.
Ukrainian television was switched off in Crimea on Thursday and replaced with Russian state channels. The streets largely belong to people who support Moscow's rule, some of whom have become increasingly aggressive in the past week, harassing journalists and occasional pro-Kiev protesters.
Part of the Crimea's 2 million population opposes Moscow's rule, including members of the region's ethnic Russian majority. The last time Crimeans were asked, in 1991, they voted narrowly for independence along with the rest of Ukraine.
"This announcement that we are already part of Russia provokes nothing but tears," said Tatyana, 41, an ethnic Russian. "With all these soldiers here, it is like we are living in a zoo. Everyone fully understands this is an occupation."