The Prime Minister rejected a claim made by Lord Ashcroft in a biography that he had "inserted a private part of his anatomy" into a dead pig's mouth at an initiation ceremony for the Piers Gaveston Society while at Oxford University.
Mr Cameron said that he stood by a denial made by close friends in recent days who said the claim was "nonsense" and "completely untrue".
The Prime Minister also suggested that Lord Ashcroft, who co-wrote the book after not being given a ministerial position by Mr Cameron, was driven by revenge.
Asked about the biography and the specific allegation concerning the pig, Mr Cameron said: "I think everybody, everyone can see why the book was written and I think everyone can see straight through it. As for the specific issue raised, you know a very specific denial was made a week ago, and I've nothing to add to that."
When the allegation surfaced last week, Downing Street said they would not "dignify" the claims with a response. However, close friends of the Prime Minister went further, saying the claims had been "nonsense" and "completely untrue".
Mr Cameron's comments are his most detailed response to the book to date. He has previously suggested Lord Ashcroft has stabbed him in the back.
Lord Ashcroft has admitted he has "beef" with Mr Cameron after being overlooked for a senior role despite giving millions of pounds to the Conservatives. The peer has claimed Mr Cameron reneged on a promise that he would get a top job should the Conservatives win the election, with reports suggesting he had expected to be made defence secretary.
Conservative whips have reportedly begun an investigation into the unnamed Tory MP who was at Oxford with Mr Cameron and said to be behind the claims.
Mark Field, the Tory MP for the Cities of London and Westminster, issued a strenuous denial he was behind the story after accusations made by senior figures appeared in the Sunday newspapers.
"I regard any insinuation that I have done this as defamatory. I categorically deny it. I didn't even know Cameron at university. I was never in the Piers Gaveston club," Mr Field said.
