The cardinal is alleged to have assaulted a priest at the Scots College in Rome in October 2003, hours after being awarded the red mitre by Pope John Paul II.
The priest, who is Scottish but now based in London, made a formal complaint to the Vatican's Congregation of Bishops last September, after which Cardinal O'Brien was summoned immediately to Rome.
The complaint, which was dealt with by Cardinal Marc Ouellet of Quebec, who was one of the early front-runners this week to become Pope, was the first which eventually led to the cleric's downfall and is not from one of the four complainers whose allegations were made public last month.
It is understood the complaint involved an attempt to grope the priest, who was known to Cardinal O'Brien. Alcohol had been consumed at an event in the Scots College attended by many priests who had travelled to Rome especially for his elevation. Scots based at the Vatican also attended.
The complaint has been erroneously reported in the media as having taken place in 2001.
The Herald understands the complaint has been taken seriously. Within a few weeks of the claims being made, the cardinal had cancelled all public engagements and stood down as president of the bishops' conference, the decision-making body for the Catholic Church in Scotland. It also coincided with a bout of ill health which led to him being hospitalised for nine days in October 2012.
Senior sources within the Catholic Church have said allegations of sexual misconduct within the Vatican, more so than any other claims he is facing, could lead to the former Archbishop of St Andrews and Edinburgh becoming only the fourth cardinal in 200 years to be stripped of the position. One source said: "If Pope Francis wasn't familiar with the problems faced by the Church in Scotland, he certainly is now."
The accusation comes just a day after another former trainee priest alleged he was abused by Cardinal O'Brien while at a seminary as a teenager.
The man, who is now in his 50s and who had known the churchman since childhood, has claimed the cardinal kissed and groped him at the unnamed seminary in the early 1980s while the pair were alone.
Now, the ex-seminarian has told how Cardinal O'Brien warned him about the repercussions for his career of revealing the incident and how over the years he has fought back the urge to confront the cardinal over the incident.
He has also told of his difficulties in the time since the revelations were made and the pain of the past 30 years when even close relatives, who remain unaware of the allegations, celebrated Cardinal O'Brien's rise through the Church ranks.
Cardinal O'Brien has retreated into privacy following claims nearly three weeks ago he "behaved inappropriately" towards three priests and a now former priest in the 1980s.
Following the revelations, he was
relieved of his post as Archbishop of St Andrews and Edinburgh and subsequently admitted his sexual conduct had "fallen below the standards expected of me as a priest, archbishop and cardinal".
It is understood the reference to his time as cardinal refers, in part, to the allegations from Rome to which Cardinal O'Brien is believed to have confessed in the aftermath of the revelations.
He became cardinal on October 21, 2003, the same day as an outside contender for the papacy, Australia's George Pell.
A spokesman for the Catholic Church in Scotland said: "A number of complaints have been passed directly to the Vatican.
"The detail and nature of them is not known to the Bishops' Conference of Scotland."
No-one from the Papal Nuncio's office in London or the Vatican press office was available for comment.