Pope Benedict XVI accepted his resignation last week, the Vatican said in a statement released Monday.
O'Brien said he submitted his resignation to the pope months ago, citing his upcoming 75th birthday and his health, according to a statement released by the Scottish Catholic Media Office.
The resignation follows a Sunday report by the British newspaper The Observer that three priests and one former priest leveled allegations against O'Brien that date back 30 years.
The final days of Benedict XVI's papacy Key moments in pope's resignation
The Observer did not recount details of the claims or identify any of O'Brien's accusers, but said one of the priests alleged "that the cardinal developed an inappropriate relationship with him."
O'Brien did not attend Mass at St. Mary's Cathedral in Edinburgh on Sunday, but the Scottish Catholic Media Office told CNN that the cardinal "contests these claims and is taking legal advice."
His accusers took their complaints to the Vatican representative in Britain and demanded O'Brien's resignation, The Observer reported. At the Vatican, Father Federico Lombardi, a spokesman for the church, told reporters that Benedict has been informed of the allegations.
The Irish-born O'Brien was scheduled to retire on St. Patrick's Day, his 75th birthday.
As late as last week, he appeared to be making plans to take part in the conclave, when the College of Cardinals gathers in Rome to pick a successor to Benedict.
The pope accepted O'Brien's resignation ahead of leaving the papacy on Thursday, becoming the first pope to step down since 1415.
"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 apologise to all whom I have offended," O'Brien said in the statement released by the Scottish Catholic Media Officer.
The accusations against O'Brien follow a buzz in Italian media about claims that gay clergy may have made themselves vulnerable to blackmail by male prostitutes, setting off speculation that a brewing scandal may have triggered Benedict's resignation.
The Vatican vehemently denied the allegations Saturday, with Secretary of State Tarcisio Bertone criticizing a rash of "often unverified, unverifiable or completely false news stories" as the cardinals prepare for their conclave.
Benedict announced his resignation on February 11, saying that at 85, he was too weak to continue his duties.
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