UEFA have upheld the decision to reject Real Madrid's request to register both Lassana Diarra and Klaas Jan Huntelaar to play in the Champions League knockout stages.
UEFA's appeals body made the decision after Madrid had tried to overturn the initial ruling made by European football's governing body last Thursday.
Madrid had been hoping to register both Diarra and Huntelaar for the Champions League even though regulations state that only one player who has already played in Europe this season can be registered.
Both Diarra and Huntelaar had played in the UEFA Cup this season, for Portsmouth and Ajax respectively, prior to joining Madrid earlier this month.
Madrid had gone to UEFA claiming they had a "different interpretation'' of the rule, but both the Spanish club's initial request and subsequent appeal have now been turned down.
A UEFA statement read: "UEFA's appeals body today upheld the decision taken by the UEFA control and disciplinary body on January 8, in accordance with article 17.18 of the [UEFA Champions League] competition regulations.
"They rejected an appeal by Real Madrid CF in relation to the Spanish club's request to be able to register two players who have already competed in UEFA competition this season, as part of their A-list squad for the knockout rounds of the UEFA Champions League.''
Madrid are now expected to take their case to the Court of Arbitration for Sport.