The head of the US Senate intelligence committee has publicly accused the CIA of improperly accessing computers used by congressional staff.
Senator Dianne Feinstein said on the Senate floor that such activities "may have undermined the constitutional framework" of government oversight.
The Senate panel was investigating allegations of abuse during a CIA detention and interrogation programme.
A CIA internal watchdog has been tasked with looking into the alleged hacking.
BBC Washington correspondent Rajini Vaidyanathan says the spat is remarkable because it is so public.
It is highly unusual for a senior US senator - and one on the intelligence committee - to level these kind of accusations on the chamber floor.
Ms Feinstein is effectively questioning the impartiality and independence of the CIA, our correspondent says.
"I am not taking it lightly," Ms Feinstein said of the matter on Tuesday, adding that the CIA may have violated federal laws in its alleged conduct.
But CIA director John Brennan rejected the Senate allegations.
"Nothing could be further from the truth," Mr Brennan said at an event in Washington on Tuesday. "The matter is being dealt with in an appropriate way, being looked at by the right authorities, and the facts will come out."
The agency is also accused of secretly removing more than 900 documents from computers used by the Senate intelligence committee during an investigation into alleged CIA abuse.
Those computers were provided by the CIA to congressional members of staff at a secure site in northern Virginia so that Senate investigators could review millions of pages of top secret documents.
The alleged CIA abuse stemmed from a detention and interrogation programme under former President George W Bush.
Ms Feinstein has previously said that the committee's 6,000-page "comprehensive review" - completed in 2013 and encompassing six million pages of records - found that the CIA programme had yielded little or no significant intelligence.
On Tuesday, the Senate intelligence committee chairwoman said such improper access to congressional networks, if true, amounted to attempted intimidation of investigators.
She also said she had requested an apology from the agency and an acknowledgment that the search was inappropriate, but had "received neither" despite sending letters to the agency requesting information on 17 and 23 January.
Ms Feinstein noted that CIA inspector general David Buckley had been tasked with looking into the alleged actions.
White House Press Secretary Jay Carney declined to comment publicly on Tuesday, but said the matter had been referred to the Department of Justice for investigation.
"Heads should roll, people should go to jail if it's true," Republican Senator Lindsay Graham, speaking on Tuesday of the allegations against the CIA, told the Associated Press news agency.
"The legislative branch should declare war on the CIA."