FIFA has rejected Leicester City's request to sign Adrien Silva from Sporting CP.
The Premier League club believed they had beaten the 11pm (BST) deadline to purchase the midfielder on August 31, although that was disputed by FIFA's Transfer Matching System.
The Foxes had agreed a deal worth up to €29.5 million to bring in the Portugal international, but the paperwork was subsequently forwarded to world football's governing body after concerns around the deadline being missed.
And after referral to the Players' Status Committee, any hopes Leicester may have had of forcing through the deal have now been dismissed, with a FIFA spokesman confirming to Sky Sports that the transfer won't be going ahead.
The statement read: "We can confirm the following decision of the Single Judge of the Players' Status Committee: the petition made by the Football Association for permission to request the International Transfer Certificate for the player Adrien Silva and subsequently register the player for its affiliated club, Leicester City FC, is rejected."
Silva was seen as a direct replacement for Danny Drinkwater, who joined Chelsea on deadline day.
The player had been in limbo over the past month, however, with his future remaining uncertain and having even been unable to train with Craig Shakespeare's side.
To add to Silva's disappointment, the 28-year-old was not included in Fernando Santos's Portugal squad for their final two World Cup qualifiers against Andorra and Switzerland.