Russia warplanes struck at rebels battling Islamic State militants, including forces backed by the United States, in southern Syria on Thursday, a senior U.S. defense official said.
The official, who spoke on condition of anonymity, criticized the Russian air strikes near al-Tanf and said no Russia or Syrian ground forces were in the area at the time.
"Russia's latest actions raise serious concern about Russian intentions," the official said.
"We will seek an explanation from Russia on why it took this action and assurances this will not happen again."
Dmitry Peskov, a Kremlin spokesman, said the mingling of so-called moderate rebels with Nusra Front fighters on the ground in Syria made it hard to distinguish between the two when it came to targeting air strikes.
"It really is complicating anti-terrorist action," he said, referring to Russia's campaign of air strikes in Syria.
British-based monitoring group the Syrian Observatory for Human Rights said warplanes had struck a meeting of U.S.-backed forces fighting against Islamic State in al-Tanf village, near the al-Tanf border crossing with Iraq, killing two fighters and wounding four others.
It said it was unclear whose planes had carried out the attack, however.
Washington has consistently refused to join forces with Russia in Syria against Islamic State ever since Moscow launched its campaign of air strikes in September last year, accusing it of acting solely to prop up Syrian President Bashar al-Assad.
The United States has called on Assad to step down.
Communication between the U.S. and Russian militaries on Syria has been limited to contacts aimed at avoiding an accidental clash as they carry out rival bombing campaigns and small numbers of U.S. forces operate on the ground.