Fife Police confirmed they were making inquiries into the latest allegation against Dunfermline MSP Bill Walker of an attack said to have taken place more than 20 years ago.
Mr Walker, who has been accused of domestic abuse over four decades by three former wives and a teenage daughter, denies the rape claim.
He has so far resisted calls by opposition parties to quit Holyrood but after the latest allegation by a woman in Edinburgh all Fife Labour MSPs yesterday demanded his resignation.
In a statement John Park, Richard Simpson, Claire Baker and Helen Eadie said: "These are highly disturbing allegations and, coming on top of what we now know about Mr Walker's past, we have serious concerns about his fitness to be an MSP. He should resign from the Scottish Parliament today."
Mr Walker, also a Fife councillor, was defended by fellow Fife SNP councillor Douglas Chapman who said the allegations were "inconsistent with the discussions I've had with Bill on these issues".
He said: "Some of these events took place 20 to 30 years ago and the moral code was very different."
He added: "In no way am I condoning violence against men or women but it was a different time."
Dunfermline and West Fife Labour MP Thomas Docherty said: "Mr Chapman should spell out exactly what he means by these remarks and apologise."
Mr Chapman said: "I condemn violence against women and domestic abuse of all kinds, regardless of when it was committed."
Mr Walker, 70, has said he intends to carry on as an independent MSP.
An SNP spokesman said: "Bill Walker was expelled from the party over a week ago, subject to an appeal against the decision."
A Tory party spokesman said Mr Walker should to the "honourable thing" and resign.
Green Party co-leader Patrick Harvie said: "Bill Walker has outstayed his welcome and should leave the Scottish Parliament."
Mr Walker could not be contacted yesterday.
MSPs and MPs can only be removed from parliament if they are jailed for a year or more.