Tory chairman Grant Shapps has resisted pressure from Eurosceptic Conservative MPs to form a pact with Ukip at the 2015 general election.
Backbenchers Douglas Carswell and Jacob Rees-Mogg said a deal with Ukip would avoid splitting the centre-right vote for the Westminster contest.
But Shapps ruled out a pact or fielding joint candidates with Nigel Farage's party, saying there was "no question" of it happening.
He told the BBC: "We are not going to have a pact or joint candidates or what have you. It can't happen anyway, it can't happen on a technical basis because we do not allow joint candidates to stand. We are a single party, no other party would have joint candidates."
He added: "We are the Conservative Party, we are the best chance of offering an in/out referendum, the only chance of having an in/out referendum. You need 326 MPs to pass an act to get that referendum. Currently Ukip have zero."
But Rees-Mogg told the BBC it was "very destructive" to the aims of conservative parties for Ukip and the Tories to fight each other.
He said: "If you look at the results that are coming through, the 'small c' conservative faction in British politics is picking up a very large share of votes.
"In a first-past-the-post system, if they don't get those votes into one pot, then both those sides end up losing."
While the Conservatives should try to win back voters from Farage's party, he added that there could be a case for candidates to fight on a joint "coupon" where they agreed.
"With the promise of a referendum, the Conservative Party and Ukip are moving in exactly the same direction, that is offering British people a final decision on whether we have continued membership of the European Union. That is surely pushing us towards a coupon of some kind."
Carswell made his call for a pact in the Daily Telegraph, claiming that Tory attacks on the "basically good people" in Ukip had backfired.
He said: "The price of not doing a deal could be to not get a referendum (on the EU) and to have Ed Miliband in Downing Street."