In a statement sent to AFP, Pentagon spokesman Peter Cook condemned the fighting south of the Syrian town of Jarabulus.
"We are closely monitoring reports of clashes south of Jarabulus - where ISIL is no longer located - between the Turkish armed forces, some opposition groups, and units that are affiliated with the SDF (Kurdish-led Syrian Democratic Forces)," he said.
ISIL is an acronym for the Islamic State (IS) group.
"We want to make clear that we find these clashes unacceptable and they are a source of deep concern."
"This is an already crowded battle space. Accordingly, we are calling on all armed actors to stand down immediately and take appropriate measures to de-conflict."
The comments come after Turkish forces began a two- pronged operation against IS and Kurdish fighters from the People's Protection Units (YPG) inside Syria on Wednesday.
The YPG is the main component of the US-backed SDF alliance, which has been fighting IS in northern Syria.
Turkey considers the YPG a "terrorist" group and said today it would continue to target the group if it failed to retreat east of the Euphrates River.
US Vice President Joe Biden made a visit to Turkey last week, during which he said Washington was looking "to preserve the territorial integrity of Syria," Cook explained.
"We have made this clear to the YPG elements of the SDF. We have reiterated our view that the YPG must cross back to the eastern side of the Euphrates and understand that has largely occurred," the Pentagon spokesman added.
"We maintain and will continue to engage our partners on the ground to ensure that our collective efforts to deal ISIL a lasting defeat are well-coordinated and synchronized."
Yesterday, dozens of people were killed in Turkish bombardments in Syria as Ankara ramped up its unprecedented offensive.
Ankara said it had killed 25 Kurdish "terrorists" and insisted the army was doing everything possible to avoid civilian casualties.
But the Britain-based Syrian Observatory for Human Rights said at least 40 civilians were killed in Turkish shelling and air strikes on two areas held by pro-Kurdish forces, the first report of significant civilian casualties in Turkey's operation.
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