Andres Iniesta has failed to recover from a calf injury and will miss Barcelona's trip to Villarreal on Sunday, the club have confirmed.
Iniesta, 33, was taken off during last week's draw with Celta Vigo and missed the midweek win against Sporting Lisbon in the Champions League.
He returned to training on Friday and Barca hoped to have him available for the game at the Estadio de la Ceramica.
However, the Catalan club confirmed on Saturday that the game will come too soon for the midfielder.
Like Iniesta, Ousmane Dembele, Rafinha and Javier Mascherano all took part in training this week but none will be available for the game against sixth-placed Villarreal.
Samuel Umtiti, who will be out for two months with a torn hamstring, is also missing.
With Umtiti and Mascherano both absent, Ernesto Valverde has called up B team defender David Costas to provide backup for Thomas Vermaelen and Gerard Pique.
However, there's no room in Barca's matchday squad for Gerard Deulofeu or Arda Turan.
It's the second straight league game Deulofeu has not been selected, while he was also an unused substitute in midweek against Sporting, despite Valverde fielding an experimental lineup.
Turan has not featured in an official game this season, but is fit and available for selection again. However, it's unlikely the Turkish international will play for the club again ahead of an expected January departure.
Barca can maintain their five-point lead atop the league with a win against Villarreal, while avoiding defeat will allow them to extend their unbeaten streak in all competitions to 23 games.