Billboards urging Nicola Sturgeon to "resign" appeared on Friday in Scotland's three largest cities after she indicated she would refuse to quit even if an inquiry found she broke the ministerial code in the Alex Salmond scandal.
The "#ResignSturgeon" message appeared on electronic advertising boards in Edinburgh, Glasgow and Aberdeen as part of a campaign by Unionist groups The Majority and Scotland Matters.
It also featured on an aerial banner flown over the Scottish Parliament building and Edinburgh city centre, and later Glasgow city centre.
The Majority said they represented "the silent majority" of Scots who are "angry and frustrated by Nicola Sturgeon’s shenanigans".
Alan Sutherland from Scotland Matters said: "We call on the First Minister to do the right thing for Scotland: resign and let us focus on recovery from the pandemic.
"She has done great damage to our country and Parliament’s reputation, here and abroad, by conducting an undignified, very public dispute with her former SNP colleague, while preventing the Salmond enquiry from seeing evidence that is crucial to a proper investigation."
The messages appeared the day after Ms Sturgeon refused to provide any guarantee that she would quit if an independent inquiry found she broke the ministerial code, despite this being the usual sanction.
She instead told MSPs that "we can debate in this chamber" what her punishment should be and made clear she will lead the SNP into May's Holyrood election, regardless of the findings.
Opposition politicians expect her to argue that the Scottish people should deliver their verdict at the ballot box rather than her following convention by quitting. Opinion polls suggest the SNP is on course to win a majority.
James Hamilton QC, an Irish lawyer, is investigating allegations she broke the code, including whether she misled parliament about when she found out about her government's investigation into sexual misconduct claims against Mr Salmond.
She is also accused of failing to inform her civil servants of a series of secret and unminuted discussions she held with Mr Salmond about the investigation, with the code stipulating that government business must be recorded.
Ms Sturgeon told parliament she had refused Mr Salmond's pleas to intervene in the case but this was contradicted by him and Duncan Hamilton, a former SNP MSP who was present during a summit at her home. In written evidence, Mr Hamilton said: "My clear recollection is that her words were 'If it comes to it, I will intervene'."
In another potential breach, it is alleged the Scottish Government persisted with its court battle against Mr Salmond's judicial review of the investigation long after receiving legal advice to concede. The SNP did not respond to a request for a comment.