Last week the Evening Times reported the land in Paisley was sold by the government for just £50,000, five years after it was purchased for £840,000.
Labour leader Johann Lamont told the First Minister the land was bought and then later sold back to businessman John McGlynn who she said was a former Tory donor and now a supporter of Yes Scotland in the referendum campaign.
She said: "Is there some connection here or has Mr McGlynn just benefitted from the First Minister's gross incompetence with public funds?"
Alex Salmond said ministers had no role in land transactions and dismissed her suggestions as "ridiculous.
He said: "If Johann Lamont has any evidence that anything untoward has been happening in property transactions, let her bring it to the chamber or into public notice."
He suggested she had no such evidence and asked her not to make attacks on business people in Scotland.
Mr McGlynn said he would be seeking a meeting with the Labour leader and called for her to make an apology. He said the dealings were in the public domain and open to scrutiny.
He said: "My company's transactions involving the ill-fated Garl project are a matter of public record and beyond reproach.
"Land which we owned was purchased for £840,000 and a compensation figure, which is the subject of a continuing and legally binding confidentiality agreement."
He added: "On a general point, I jealously protect my reputation and comments have been made in the chamber which I find ill-founded and objectionable in the extreme.