Deputy leader of the Commons Nigel Griffiths has quit the government in protest at plans to renew the UK's Trident nuclear weapons system.
He resigned "with a heavy heart but a clear conscience" ahead of Wednesday's House of Commons vote on the plan.
Although a Labour rebellion is expected the Tories back renewal of Trident, making a government defeat unlikely.
Jim Devine, a parliamentary private secretary, has also indicated he will resign over the issue.
In a statement after handing his letter in to Number 10 Downing Street, Mr Griffiths, MP for Edinburgh South, said: "I'm resigning with a heavy heart but a clear conscience.
"I intend to make a personal statement in the House of Commons to colleagues and it is only right that they hear the reasons first."
Tory backing
MPs will debate and vote on Wednesday evening on the £20bn plan to replace submarine-based Trident nuclear weapons system.
Ministers say the long lead time in developing and building the replacement submarines means a decision needs to be taken soon on replacing Trident.
"I hope other MPs will follow suit and start leading the UK down the path of nuclear disarmament"
Conservative leader David Cameron made clear his support for the plans on BBC Radio 4's Today programme on Monday.
He said: "I think it needs to be done and I've always supported Britain having a nuclear deterrent, so when Trident comes to the end of its life it needs to be replaced."
The Liberal Democrats have said Parliament should not make its decision until at least 2012.
Mr Griffiths, an MP since 1987 and deputy Commons leader since 2005, previously served as a trade and industry minister.
Survey
Commons leader Jack Straw paid tribute to him in the Commons on Monday, saying: "I would like to place on record my appreciation for the excellent work which he undertook in this place."
Labour MP Jeremy Corbyn, an opponent of Trident renewal, said: "I hope other MPs will follow suit and start leading the UK down the path of nuclear disarmament, not re-armament, on Wednesday."
Alan Mackinnon, chairman of Scottish CND, said: "We welcome the principled stand taken by Griffiths on this crucial issue and we hope his example will be followed by other Labour MPs."
A survey for BBC Radio 4's The World This Weekend programme found that out of the 101 Labour MPs who responded, 22 said they supported the renewal of Trident.
A total of 64 said they opposed it, and a further 15 remained undecided.
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