The move follows allegations – which he contests – of inappropriate behavior towards priests dating from the 1980s.
In a statement, Cardinal Keith O’Brien apologized to those he had offended during his ministry.
The cardinal confirmed he would not take part in the election for a successor to Pope Benedict XVI – leaving Britain unrepresented in the election.
The Vatican confirmed that the cardinal has stepped down from his post.
Cardinal Keith O’Brien said in his statement he had already tendered his resignation as Archbishop of St Andrews and Edinburgh, due to take effect when he turned 75 next month, but Pope Benedict “has now decided that my resignation will take effect today”.
He said the pontiff would appoint an apostolic administrator to govern the archdiocese in his place until his successor is appointed.
The cardinal also said: “I have valued the opportunity of serving the people of Scotland and overseas in various ways since becoming a priest.
“Looking back over my years of ministry: For any good I have been able to do, I thank God. For any failures, I apologize to all whom I have offended.
“I do not wish media attention in Rome to be focused on me – but rather on Pope Benedict XVI and on his successor.
“However, I will pray with them and for them that, enlightened by the Holy Spirit, they will make the correct choice for the future good of the Church.”
Cardinal Keith O’Brien’s role as Britain’s only representative in the papal election next month would have been one of his last acts before he retired.
The Observer reported that the three priests and one former priest – from the diocese of St Andrews and Edinburgh – complained to the Pope’s representative to Britain, nuncio Antonio Mennini, in the week before February 11, when Pope Benedict announced his resignation, of what they claimed was the cardinal’s inappropriate behavior towards them in the 1980s.
The former priest claims Cardinal Keith O’Brien made an inappropriate approach to him in 1980, after night prayers, when he was a seminarian at St Andrew’s College, Drygrange.
The complainant says he resigned as a priest when Cardinal Keith O’Brien was first made a bishop.
A second statement from another complainant says he was living in a parish when he was visited by Keith O’Brien, and inappropriate contact took place between them.
A third complainant alleges dealing with what he describes as “unwanted behavior” by the cardinal in the 1980s after some late-night drinking.
And the fourth complainant claims the cardinal used night prayers as an excuse for inappropriate contact.
Scotland’s First Minister Alex Salmond said: “It would be a great pity if a lifetime of positive work was lost from comment in the circumstances of his resignation.
“None of us know the outcome of the investigation into the claims made against him but I have found him to be a good man for his church and country.”
Jack Valero, from Catholic Voices, a media lobby group that represents many Catholics in the UK, says the allegations against the cardinal have been handled properly.
“I think it’s right that he’s resigned, faced with these allegations.
“I am very happy that they have been taken seriously, that the nuncio – who is the Pope’s representative in the UK – has written to the four people who have made the allegations to thank them for speaking out, and that the whole thing has been done so quickly. I think this shows a new spirit.”
Colin MacFarlane, director of gay rights group Stonewall Scotland, which last year named the cardinal as Bigot of the Year said: “We trust there will be a full investigation into the serious allegations made against Cardinal Keith O’Brien.
“We hope that his successor will show a little more Christian charity towards openly gay people than the cardinal did himself.”
Clifford Longley, a religious commentator and columnist for the Catholic newspaper The Tablet, said the cardinal’s resignation was “devastating”.
“The worst thing that could possibly have happened to the church at this moment is to have another row like this when there already so many going on,” he said.
Cardinal Keith O’Brien missed celebrating Sunday Mass in St Mary’s Cathedral in Edinburgh, which marked Pope Benedict’s eight years in office, ahead of the pontiff stepping down this week.
Last week Cardinal Keith O’Brien said he believed priests should be able to marry if they wished to do so, saying the new Pope could consider whether the Roman Catholic Church should change its stance on some issues not of divine origin.
Cardinal Keith O’Brien:
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