Gretna have lost their battle for survival and resigned from the Scottish Football League.
The administrator running the troubled club concluded that Gretna could not continue running as a business.
Gretna only entered the SFL structure in 2002 and completed an astonishing rise to the Premier League in 2007.
However, the club ran into financial problems when benefactor Brooks Mileson withdrew his support and they were relegated from the top flight.
Gretna went into administration in March with debts totalling about £4m.
They were immediately penalised 10 points by the SPL and 22 players were made redundant, ensuring the already struggling team would be relegated.
The administrator called in was only able to see Gretna through to the end of the season after an advance of prize money from the SPL.
With doubts over the club's ability to fulfil fixtures next season, the SFL took the decision to drop Gretna down to Division Three.
But the one previously interested party withdrew their offer to purchase the club and Gretna are now out of business altogether.
An SFL spokesman said: "I have received intimation from the Gretna FC administrator of their resignation from the Scottish Football League with immediate effect.
"I have accepted this resignation on behalf of the Scottish Football League and accordingly, we now have a vacancy in the Third Division and are seeking applications."
The SFL have set a deadline of 12 June for prospective new clubs to apply for membership.
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The SFL Management Committee will then visit each applicant club by 27 June.
Applicant clubs will then be invited, following assessment, to present their business and football plans to all 29 members of the SFL at a Special General Meeting to be held at Hampden Park on 3 July.
The 29 SFL clubs will then vote on the day to decide which club has been invited to join the SFL as a Third Division club with effect from the 2008-09 season.
Spartans, Annan Athletic, Preston Athletic and Cove Rangers are all preparing applications.
Bookmaker William Hill has made Spartans 5/6 favourites to replace Gretna in the Scottish Football League, while Cove Rangers are 11/4, Annan Athletic 7/2 and Preston Athletic outsiders at 5/1.
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