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"I want to make it absolutely clear to Assad and those under his command: The world is watching," Obama said in a speech to a gathering of biological, chemical and nuclear weapons proliferation experts.
"The use of chemical weapons is and would be totally unacceptable and if you make the tragic mistake of using these weapons, there will be consequences and you will be held accountable," Obama said.
He did not say how the United States might respond, but White House spokesman Jay Carney said earlier that "contingency planning" was under way when asked whether the use of military force was an option.
It was unclear what has motivated US officials to think that Syria might be on the verge of using chemical weapons.
An American official said the United States is concerned that Syria may be preparing to combine the chemicals needed to make sarin gas.
As Assad's government has shown signs of increasing strain in response to recent advances made by the rebels, Carney said the United States has grown concerned that the Syrian president might be considering the use of chemical weapons.
This would, Carney said, "cross a red line for the United States."
Some US Republicans have been critical of the Obama administration's response to the Syrian crisis as thousands of people have been killed during the country's civil war.
During the presidential campaign, Obama's Republican challenger, Mitt Romney, said the United States should facilitate the arming of Syrian rebels, a step Obama has not taken.
Obama said in his speech on Monday that the United States would continue to support the legitimate aspirations of the Syrian people, engaging with the opposition and providing them with humanitarian aid. He said his goal is a transition in Syria to a country that is free of Assad.