"I can confirm that the US struck a vehicle killing several Al-Qaeda militants," said spokesman Matthew Allen. "The results of this strike are still being assessed."
Read more from our special coverage on "US"
The latest strike was carried out in northwestern Syria, according to a Defense department official who asked not to be named.
The Washington Post reported the latest raid was late Tuesday.
On Monday, the Pentagon said the US military conducted an air raid on an Al-Nusra meeting in northwest Syria the previous day.
Al-Qaeda's Syrian affiliate confirmed yesterday the death of its spokesman Abu Firas al-Suri in a US air strike.
US strikes on the Al-Nusra Front in Syria have not been very frequent with their raids overwhelmingly targeting the Islamic State group.
News of the strikes came as talks in Geneva aimed at ending the conflict loomed on April 11.
Syrian peace talks which fail to address the question of President Bashar al-Assad's fate are "doomed to failure", a spokesman for the main opposition grouping involved in negotiations said.
Riad Naasan Agha, of the Riyadh-based High Negotiations Committee, said that the talks which are set to resume must focus on the future of the Syrian leader.
"If negotiations did not address the fate of Assad, it would be a waste of time and doomed to failure," he said late Tuesday at a forum hosted by Al-Jazeera in Qatar.
The UN has said the upcoming round of talks will focus on plans for a political transition to lead Syria out of five years of brutal civil war.
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