The strike on the convoy heading toward a coalition garrison near the Jordanian border was "necessitated by offensive movement with offensive capability of what we believe were Iranian-directed forces", Mattis said, adding he was not sure if there were Iranians on the ground.
Yesterday's strike occurred inside an established "deconfliction zone" northwest of the At-Tanf garrison, where British and US commandos have been training and advising local forces fighting the Islamic State group.
Such zones are agreed upon between Russia and the coalition, and are designed to stop either side inadvertently striking the other's forces on the ground and in the air.
It appeared the convoy had moved into the area against the advice of the Russians, Mattis said.
"It looks like the Russians tried to dissuade them," he said.
The Pentagon has stressed the attack did not signal broader US involvement in Syria's civil war, but Damascus today condemned the strike on pro-government forces as a "brazen attack" and said it would "not be intimidated".
An array of regular and irregular forces are battling alongside the government against rebels, including Russian and Iranian soldiers and militants from Iraq and Lebanon's Hezbollah group.
Yesterday's strike comes in the context of growing tension over which forces will take on IS in Syria's east.
President Bashar al-Assad's army is trying to prevent US-backed forces from leading that fight.
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