Serie A teams who still wish to pursue the European Super League on June 21 will lose their league membership, according to a new ruling in Italy.
Italian giants Juventus, Inter and Milan signed up to the controversial breakaway competition earlier this month among 12 elite European clubs.
Proposals guaranteed participation for the dozen founding teams, who would no longer enter the UEFA Champions League.
But the anti-competitive tournament prompted outrage around the football world, and pressure from fans, players, coaches, governing bodies, governments and the media soon told.
The Premier League's 'big six' all backed out within two days of the Super League's launch, while Inter quickly followed.
Milan appeared to distance themselves from the new competition, too, but Juventus, while acknowledging the existing format cannot work, retain hope of reform in European football.
Juve chairman Andrea Agnelli has been credited with playing a key role in the organisation of the tournament.
But initial plans suggested the Bianconeri and their allies would continue to play in domestic competitions while contesting the Super League.
A new regulation, passed on Tuesday (AEST), means this cannot happen.
Italian Football Association (FIGC) president Gabriele Gravina said: "Those who believe they should participate in a competition not authorised by the FIGC, UEFA and FIFA lose membership.
"At the moment, we have no news of who remained and who left the Super League.
"This rule refers to national licenses. It is clear that if, on June 21, the deadline for registration applications, someone wishes to participate in competitions of a private nature, they will not take part in our championship."
Juve would appear to be the club at most serious risk, although Real Madrid president Florentino Perez has claimed Milan are also still involved.
Both Juve and Milan first have work to do to clinch qualification for the Champions League, the tournament they sought to break away from.
Andrea Pirlo's team were held to a 1-1 draw at Fiorentina on Monday and are fourth, level on points with fifth-placed Milan, who were thumped 3-0 by Lazio on Tuesday.