The Pentagon has said unilateral military action in north-east Syria by any party would be "unacceptable", in a warning after Turkey said it would launch a new military operation in the region within days to target Kurdish militia fighters.
Ankara and Washington have long been at odds over Syria, where the United States has backed the YPG Kurdish militia in the fight against ISIS militants.
Turkey says the YPG is a terrorist organisation and an extension of the outlawed Kurdistan Workers' Party (PKK), which has waged an insurgency in south-eastern Turkey for 34 years.
Within hours of Turkish President RecepTayyip Erdogan's announcement on Wednesday of a planned operation against the Syrian Kurdish militia, the US defence department said such action would undermine the shared interest of securing the border between Syria and Turkey in a sustainable way.
"Unilateral military action into north-east Syria by any party, particularly as US personnel may be present or in the vicinity, is of grave concern," Pentagon spokesman Sean Robertson said.
"We would find any such actions unacceptable."
Turkey has launched military campaigns over the past two years to sweep YPG fighters from territory west of the Euphrates, but has not gone east of the river - partly to avoid direct confrontation with US forces.
But Mr Erdogan's patience with Washington over Syria - specifically a deal to clear the YPG from the town of Manbij, just west of the Euphrates - seems to have worn thin.
"We will start the operation to clear the east of the Euphrates from separatist terrorists in a few days. Our target is never US soldiers," Mr Erdogan said at a defence industry summit in Ankara.
"This step will allow for the path to a political solution to be opened and for healthier co-operation."
Turkey has repeatedly voiced frustration about the speed of implementation of the Manbij deal, saying last month that the agreement should be fully carried out by the end of this year.
The Pentagon said co-ordination and consultation between the US and Turkey was the only way to address security concerns and that Washington was focused on working closely with Ankara.
"We believe this dialogue is the only way to secure the border area in a sustainable manner, and believe that unco-ordinated military operations will undermine that shared interest," Commander Robertson said.
He added that while the US was fully committed to Turkey's border security, it also remained committed to working with the Syrian Democratic Forces (SDF), which includes Kurdish YPG fighters, to defeat ISIS.
Turkish and US troops began joint patrols near Manbij last month, but that co-operation has been complicated by Turkey shelling Kurdish fighters to the east of the Euphrates.
Earlier this year, the SDF temporarily suspended an offensive against ISIS after Turkish shelling in northern Syria.
The Pentagon says it has about 2,000 troops in Syria.
Last month, the US said it would set up observation posts on the border between Kurdish-held northern Syria and Turkey after Turkish cross-border shelling killed four Kurdish fighters.
Three observation posts have now been set up, a US official told Reuters on Wednesday. The official said the positions were clearly marked and any force attacking them "would definitely know they are attacking the United States".
Turkish officials held talks in Ankara this week with the US special representative for Syria engagement, Jim Jeffrey. An SDF military source said Mr Jeffrey met the force's leadership in northern Syria on Wednesday.
Mr Erdogan said Turkey was the victim of a "stalling tactic" over Manbij and that ISIS no longer posed a threat in Syria.
"Now, it's time to realise our decision to disperse the circles of terror east of the Euphrates. The fact that we have deep differences in perception with the United States is no secret," he said.