Diego Costa was left out of the Spain squad announced by coach Luis Enrique on Thursday, ahead of games against Wales and England.
Costa sustained a thigh strain playing for Atletico Madrid against Club Brugge in the Champions League on Wednesday. Isco, Dani Carvajal, Inigo Martinez and Sergi Roberto are also injured.
"I really regret losing players but it gives other players an opportunity to show me their level," Luis Enrique said.
Wolves defender Jonny Otto, on loan from Atletico, received his first international call-up while there were returns to the squad for Real Betis defender Marc Bartra, Borussia Dortmund forward Paco Alcacer, who is on loan from Barcelona, and Atletico Madrid's Koke.
Koke was not picked for the games against Croatia and Spain last month but has been in fine form for Atletico.
"He deserves to be here," Luis Enrique said. "The doors to the squad are always open."
Alvaro Morata was also included despite failing to score for Chelsea since the last international break.
Barcelona defender Jordi Alba, a surprise absentee from Luis Enrique's first squad, was again left out. Luis Enrique denied Alba's exclusion was due to any personal disagreement dating back to his time in charge of Barcelona.
"Personal reasons do not come into it for me," Luis Enrique said. "I have done it for professional reasons, there are many players out of this list who deserve to be in."
Spain play Wales in a friendly in Cardiff on October 11 before facing England in the UEFA Nations League on October 15 in Seville.
Goalkeepers: David De Gea (Manchester United/ENG), Kepa Arrizabalaga (Chelsea/ENG), Pau Lopez (Real Betis)
Defenders: Cesar Azpilicueta (Chelsea/ENG), Raul Albiol (Napoli/ITA), Nacho, Sergio Ramos (Real Madrid), Marcos Alonso (Chelsea/ENG), Gaya (Valencia), Marc Bartra (Real Betis), Jonny Otto (Wolverhampton/ENG)
Midfielders: Sergio Busquets (Barcelona), Rodrigo, Saul, Koke (Atletico Madrid), Thiago Alcantara (Bayern Munich/GER), Dani Ceballos (Real Madrid)
Forwards: Marco Asensio (Real Madrid), Alvaro Morata (Chelsea/ENG), Suso (AC Milan/ITA), Rodrigo Moreno (Valencia), Iago Aspas (Celta Vigo), Paco Alcacer (Borussia Dortmund/GER)