The Tories have written to Tony Blair urging him to order an inquiry into whether John Prescott broke ministerial rules by visiting a US billionaire.
Mr Prescott still faces questions about a possible conflict of interest
The letter follows Culture Secretary Tessa Jowell's claim that she discussed casino policy with Mr Prescott.
Mr Prescott has been criticised by the Commons standards committee for failing to declare hospitality during the trip to Philip Anschutz's Colorado ranch.
The tycoon wants to build a super casino to be at the Millennium Dome.
The letter, from Conservative shadow culture spokesman Hugo Swire, was prompted by Ms Jowell's evidence to the Commons culture select committee.
The culture secretary said she could not recall any meetings with the deputy prime minister regarding licence applications, but did remember talking in general terms about regeneration policy.
A spokesman for the culture department later said Ms Jowell had spoken to Mr Prescott in December 2004 "on the periphery" of another meeting, ahead of the publication of a government policy statement on casinos.
Ms Jowell had wanted to "ensure the government maximised the regeneration aspects of the new casinos," said the spokesman.
And such conversations were a "perfectly proper practice where policy issues cut across government departments".
In his letter, Mr Swire said: "Mr Prescott has always hidden behind claims that he wasn't involved in casino policy.
"Yet Tessa Jowell today admitted to the culture, media and sport select committee that he was holding meetings with her to discuss casinos and regeneration.
"This clearly shows that Mr Prescott had a role in casino policy and surely calls into question the decision to accept gifts from the American billionaire Mr Anschutz.
"Clearly John Prescott was heavily involved in government policy on issues that would directly affect Mr Anschutz's bid.
"Given today's revelation, and the mounting evidence against Mr Prescott, why won't the prime minister allow an independent investigation to take place?"
Mr Prescott has strongly denied suggestions there was anything corrupt in his friendship with Mr Anschutz.
Last week's standards committee report said the deputy prime minister had initially broken the ministerial code by accepting the tycoon's hospitality.
The fact that he later declared the visit - after coming to accept he was in breach of the rules - meant the standards committee could not take any action against him.
But standards watchdog Sir Philip Mawer said the "key question" of whether Mr Prescott should have accepted Mr Anschutz's hospitality in the first place could not be investigated under current rules.
Such an investigation can only be ordered by the prime minister, who has so far refused to do so, saying he had seen no evidence the ministerial code has been broken.
In his letter to Mr Blair, Mr Swire says that in light of Ms Jowell's comments it was now "simply untenable" for Mr Blair not to allow a full investigation to take place.
'Nothing to add'
He urges the prime minister to instruct Sir John Bourn, the prime minister's adviser on conflicts of interest, "to investigate such an inquiry as a matter of the utmost urgency".
"A failure to do so will only serve to further undermine the ministerial code and public confidence in the integrity of your government," he said.
The Liberal Democrats have already called for an independent inquiry.
Later, the prime minister's official spokesman said he had "nothing new to say" on the matter, adding: "John Prescott was not involved in planning and not involved in deciding gambling licences."