Lord Levy, the prime minister's chief fundraiser and personal envoy to the Middle East, was arrested yesterday on suspicion of perverting the course of justice.
It is the second time the peer has been arrested by police investigating the cash-for-peerages row - last July, he was arrested in connection with the sale of honours.
He was not charged with any offence and was released on bail. Last night his spokesman said he "completely denies any allegations of wrongdoing whatsoever".
The arrest comes just two weeks after another of Tony Blair's key aides, Ruth Turner, was arrested in connection with the sale of honours and on suspicion of perverting the course of justice. She has also protested her innocence.
It raises the prospect that the prime minister may be questioned by Scotland Yard for a second time - in December he was quizzed for two hours at Downing Street. However, his official spokesman told reporters today that "nothing had changed" on this issue.
The spokesman refused to comment on yesterday's arrest, but said Lord Levy remained Mr Blair's envoy to the Middle East. Questioned during prime minister's questions this lunchtime, Mr Blair also said he would not comment on an ongoing inquiry.
However, the fact that two key aides to Mr Blair have been arrested over allegations of perverting the course of justice - a very serious offence - prompted cries of a cover-up.
Last week, Downing Street rejected claims that police were looking at a second, secret email system at No 10 from which some emails may have been deleted. And today the prime minister's spokesman said it had cooperated fully with police at all times.
But in the Commons this lunchtime, SNP leader Alex Salmond asked if there was a "cover-up", and last night Ed Davey, chief of staff to Liberal Democrat leader Menzies Campbell, warned: "Increasingly this sorry affair has the whiff of Watergate about it.
"For Nixon the crime was the cover up, but we must await the result of the police investigation to see whether something similar is now happening in Downing Street."
Asked about the arrest in a BBC Scotland interview, Gordon Brown said it would be inappropriate for him to comment and urged people to "let the investigation take its course".
Questioned about claims of a cover-up, the chancellor replied: "I think the police investigation has got to be completed then people will see the full facts and I believe when people see the full facts then they will be satisfied."
Lord Levy is one of four people to be arrested as part of the investigation into claims that political parties offered wealthy backers seats in the House of Lords in return for loans. About 90 people from both the Conservatives and Labour have also been questioned.