Iran has conducted air strikes against Islamic State (IS) targets in eastern Iraq during recent days, a Pentagon spokesman says.
Rear Adm John Kirby said the US, which has conducted its own air strikes in Iraq, was not co-ordinating with Iran.
A senior Iranian military official also dismissed talk of co-operation between the two countries.
A US-led coalition has launched hundreds of air strikes against IS since August.
The US has said it would be inappropriate for Iran to join that coalition, even though the two long-time adversaries face a common enemy in IS.
Since the Islamic revolution in 1979, the US and Iran have had a fraught relationship.
Washington severed ties the following year after Iranian students occupied the US embassy in Tehran and took 52 Americans hostage.‘Nothing changed'
Rear Adm Kirby’s comments followed reports that American-made F4 Phantom jets from the Iranian air force had been targeting IS positions in the eastern Iraqi province of Diyala.
“We have indications that they did indeed fly air strikes with F-4 Phantoms in the past several days," he said.
It was up to Iraq to oversee and co-ordinate flights by different countries in its airspace, he added.
"We are flying missions over Iraq, we co-ordinate with the Iraqi government as we conduct those," he said. “ t's up to the Iraqi government to deconflict that airspace."
“Nothing has changed about our policy of not co-ordinating military activity with the Iranians."
The Deputy Chief of Staff of Iran’s Armed Forces Brig-Gen Massoud Jazayeri also denied collaboration.
He said Iran considered the US responsible for Iraq’s "unrest and problems", adding that the US would "definitely not have a place in the future of that country".
Shia-ruled Iran has close ties to Iraq’s Shia-led government, which has struggled to counter IS militants as they seized swathes of territory in eastern Syria and northern and western Iraq.
Military analysts said earlier this year that Iran was supplying Iraq with Sukhoi Su-25 attack jets to help in the fight against IS.
Shia militias trained and funded by Iran have also been sent to Iraq to support Kurdish fighters battling IS militants.