Madrid has raised the issue of secret CIA flights using Spanish airports with US officials, Washington has confirmed.
Spain said the allegations if true could damage ties with the US
The State Department said the matter was brought up with Assistant Secretary of State Dan Fried during his visit to Spain this week.
Spain is investigating reports that CIA planes carrying terror suspects made secret stopovers on its soil.
Interior Minister Jose Antonio Alonso said if the claims were true they could damage relations with Washington.
It is alleged that at least 10 CIA planes landed at airports in Majorca and the Canary Islands between January 2004 and 2005.
The CIA's controversial "extraordinary rendition" programme involves removing suspects without court approval to third party countries for interrogation.
The state department has refused to comment on specific allegations, says the BBC's Jonathan Beale in Washington.
He says the department also refused to confirm or deny claims that the CIA has set up secret prisons across the globe to where prisoners may have been transported.
Former Prime Minister Jose Maria Aznar, who backed the US war of terror and was in power when the flights allegedly started, also denied any knowledge of the stopovers.
The US continues to play down the speculation and insists that this is not an issue affecting diplomatic relations, says our correspondent.
Spain's socialist government had said it was unaware of the flights.
But Mr Alonso said if the claims were confirmed, it could damage relations with the US because such flights would be intolerable in every sense.
The UN's special representative on torture, Manfred Nowak, has called on the European Union to investigate the allegations.
Meanwhile, Sweden has also said it will launch an investigation into whether CIA prisoner flights have used its airports.