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Spain swooped to help the group of mainly sub-Saharan Africans on board the Aquarius last week, offering the charity-run ship a berth 700 nautical miles away after Italy and Malta refused to let it dock.
An Italian coast guard ship that took aboard some of the Aquarius's passengers to make the trip safer arrived soon after dawn in the eastern Spanish port, where a staff of 2,320, including volunteers, translators and health officials, awaited.
Uniformed police looked on as officials in white overalls and protective masks greeted the first migrants to step off the boat.
The Aquarius itself, run by a Franco-German charity, is expected around 0700 GMT, with some of the remaining 106 passengers aboard.
The ship's predicament gave Italy's new government the chance to assert its anti-immigrant credentials, and Spanish Prime Minister Pedro Sanchez, in office for just over a week, took the opportunity to underline a more liberal stance.
But the plight of the Aquarius has highlighted the European Union's failure to agree on how to manage huge numbers of people fleeing poverty and conflict.
"People are coming to Europe seeking European values of solidarity and support," Red Cross Secretary General Elhadj As Sy told a news conference in Valencia on Saturday.
"Anything less than that is a betrayal (of) Europe itself."
Upon arrival, the seven pregnant women on board the boats are to be immediately taken for check-ups, and everyone on board, including 123 minors, will receive psychological help, Spanish Red Cross officials said.
Anti-migrant feeling has surged in Italy, where more than 600,000 people have arrived on its shores over the past five years, helping to propel the nationalist League into a coalition government.
Far fewer come to Spain, but the numbers are rising fast.
Most Spaniards support the idea of welcoming and helping to integrate refugees, pollsters say, allowing Sanchez, a socialist, to offer migrant-friendly policies to voters who feel previous governments did not do enough.
France, which chided Italy for turning away the Aquarius, has offered to take in any passengers who qualify for asylum and want to go there.
The final boat in the convoy is due around 1000 GMT.