Diego Costa is in line for his Spain debut after being named in the squad to face Italy, but there is no place for David Villa or Juan Mata.
Atletico Madrid striker Costa declared his intention to play for Spain rather than Brazil, the country of his birth, last year.
He was subsequently included in Vicente del Bosque's party to face South Africa in a friendly in November, but missed out due to injury.
However, Costa, who has scored 27 goals in all competitions this season, is now set to make his Spain bow when Italy visit Atletico's ground the Vicente Calderon on Wednesday.
Costa is joined in the squad by the likes of Jordi Alba, Cesar Azpilicueta, Thiago Alcantara, Juanfran and David Silva - all of whom missed out against South Africa - as Del Bosque assesses his options ahead of his team's FIFA World Cup defence in Brazil.
Villa, Spain's all-time record goalscorer, is omitted, though, while Juan Mata's big-money move to Manchester United has not been enough to keep him in the squad.
Chelsea striker Fernando Torres also continues to be out of favour.
However, Del Bosque insisted players not included this squad are still in contention to travel to Brazil.
"This is just a squad to play Italy, not the World Cup," he said. "Costa is there on merit for his form with Atletico Madrid.
"Some players not on this list have a lot of merit. I appreciate many that aren't here, there's a lot of competition."
Squad in full:
Goalkeepers: Iker Casillas (Real Madrid), Pepe Reina (Napoli), Victor Valdes (Barcelona).
Defenders: Jordi Alba (Barcelona), Raul Albiol (Napoli), Cesar Azpilicueta (Chelsea), Juanfran (Atletico Madrid), Javi Martinez (Bayern Munich), Sergio Ramos (Real Madrid).
Midfielders: Thiago Alcantara (Bayern Munich), Xabi Alonso (Real Madrid), Sergio Busquets, Cesc Fabregas, Andres Iniesta, Xavi Hernandez (all Barcelona), Koke (Atletico Madrid), Jesus Navas (Manchester City).
Forwards: Diego Costa (Atletico Madrid), Alvaro Negredo (Manchester City), Pedro (Barcelona), David Silva (Manchester City).