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US Defence Secretary Leon Panetta says a video which appears to show US Marines urinating on the corpses of Afghans is "utterly deplorable".
Those who had taken part in the incident would be held accountable "to the fullest extent", he said.
The video, which was posted online, purports to show four US Marines standing over the bodies of several Taliban fighters, at least one of whom is covered in blood.
The origin of the video is not known.
Mr Panetta has ordered the commander of US and Nato forces in Afghanistan, General John Allen, to investigate the incident.
In a statement, Mr Panetta said he had seen the footage, and the Pentagon confirmed that he had spoken by telephone with Afghan President Hamid Karzai.
Earlier, Mr Karzai condemned the attacks in strong terms.
"I find the behaviour depicted in it utterly deplorable. This conduct is entirely inappropriate for members of the United States military."
The defence secretary's statement is the latest in a round of denunciations since the video emerged.
The Pentagon says it is checking the authenticity of the video, but there is nothing to indicate that the film is not genuine.
Mr Panetta did not dispute the authenticity.
In a separate news conference, Secretary of State Hillary Clinton expressed her "total dismay" at the video.
Mrs Clinton said she shared Mr Panetta's view that such behaviour was inconsistent with the standards the "that vast, vast majority of our personnel - particularly our marines - hold themselves to".
The video did not change the tone of US efforts to secure Afghanistan, she said, saying the US continued to support security and reconciliation efforts that were "Afghan-led and Afghan-owned".
Tentative moves are being made towards peace in the country, ahead of the planned withdrawal of international combat forces by the end of 2014.
The US has about 20,000 Marines deployed in Afghanistan, based mostly in Kandahar and Helmand provinces. In total, about 90,000 US troops are on the ground in Afghanistan.
The Taliban said last week that they were working to set up a political office, possibly in Qatar, that would help to facilitate negotiations with the Afghan government and Nato countries.
The video has not yet been circulated widely in Afghanistan, but there are fears that it could provoke further violence against international forces.
A statement from the International Security Assistance Force (Isaf) in Kabul said the behaviour "dishonours the sacrifices and core values of every service member representing the fifty nations of the coalition."
Earlier, Afghan President Karzai's office said: "The government of Afghanistan is deeply disturbed by a video that shows American soldiers desecrating dead bodies of three Afghans.
"This act by American soldiers is simply inhuman and condemnable in the strongest possible terms. We expressly ask the US government to urgently investigate the video and apply the most severe punishment to anyone found guilty in this crime."
Taliban spokesman Qari Yousuf Ahmadi told the BBC that this was not the first time Americans had carried out such a "wild action" and that Taliban attacks on the Americans would continue.
But a different Taliban spokesman, Zabihullah Mujahid, said the video "is not a political process, so the video will not harm our talks and prisoner exchange because they are at the preliminary stage".
Afghan Member of Parliament Fawzia Kofi said ordinary Afghans, no matter how they felt about the Taliban, would be upset by the video.
"It's a matter of a human being, respect to a human being," she told the BBC.
"I believe that the brutal acts that the Taliban did here during their government and even now is condemned by Afghans. So is watching a brutal act by international forces. We condemn that as well," she added.
The Taliban are known for applying a ruthless brand of Islamic Sharia law in areas they control and have carried out many suicide bombings and attacks which have killed civilians.