Cabinet minister Chris Grayling has delivered a damning assessment of the European Union, claiming the current terms of membership are "disastrous" for the UK.
The Commons Leader, a prominent Eurosceptic, warned that Brussels was heading towards closer union between members states, "a path that the UK will not and should not follow".
David Cameron has allowed ministers to campaign on either side ahead of the promised referendum, but not until his negotiation process with fellow EU leaders is complete, and in a carefully-worded article Mr Grayling stopped short of saying Britain should vote to leave.
But Mr Grayling's article in the Daily Telegraph set out in clear terms that he did not believe the current relationship with Brussels should continue.
"It is time for Britain to vote again on its future in Europe. But I also strongly believe that David Cameron is right to seek new terms for our membership before that vote happens," he wrote.
"I am someone who believes that simply staying in the EU with our current terms of membership unchanged would be disastrous for Britain. That's why I have always believed that it is imperative that his renegotiation takes place and delivers as much potential change as possible. It is in the interests of all Eurosceptics and of our country."
Mr Cameron set out the rules governing ministers' conduct ahead of the promised vote on the UK's membership of the EU in a minute circulated this week.
A senior source said Number 10 was "completely relaxed" about the content of Mr Grayling's article.
The Prime Minister hopes to secure a new deal for the UK in Brussels in February which will allow him to recommend a vote to remain in the EU and Mr Grayling dismissed suggestions that Mr Cameron would have to resign if he lost the referendum vote.
"The idea that a vote for leave means he has to go is simply wrong – and no more logical than suggesting that a vote to remain would mean all Eurosceptics had to quit," he said.
The Prime Minister has always insisted he has not ruled anything out if he fails to secure an agreement on his reform demands, but former foreign secretary William Hague, now a Tory peer, said he believed it was unlikely that Mr Cameron would recommend a vote to sever ties with Brussels.
He told ITV News: "It would make a huge difference to all of us if the PM came back and said these negotiations have all failed and I'm not recommending staying in the EU. Many of us would have to re-evaluate our positions. I don't think that will happen.
"On the assumption that there is a reasonable success and negotiations now taking place, and the PM says we should stay in the EU, then that is the way I would go."
Lord Hague added: "I am a long-standing critic of the EU but you have to think in 2016, a time of great economic and security and other challenges for the Western world, is it actually the right thing for the United Kingdom to leave? That's the question we're going to have to answer in a referendum.
"Would it be in our own interests to leave? That makes me worried. If what then happened was the EU became a lot weaker and the UK disintegrated then what would we have achieved by leaving the EU? There is a risk of that.
"There will be another referendum in Scotland and it would come quicker if we left the EU and Scottish nationalists would say, if you want to stay in the EU in Scotland, the way to do that is leave the United Kingdom."
One of the main stumbling blocks the Prime Minister faces is his proposal to block EU migrants claiming in-work benefits in the UK until they have been in the country for four years.
Lord Hague said alternatives may have to be considered but the PM would not "abandon" the demand for change: "I'm sure if the proposal on migration, on welfare benefits, isn't acceptable to other countries in the EU, there has to be another discussion about alternative proposals.
"Some other way so people in Britain can be sure that our welfare system is fair and is not going to be abused by people from other countries. There are different ways that you can do that. That is not going to be a subject that the PM is going to abandon altogether."