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).
|Coach: Del Bosque|
for Fàbregas 46'
for Casillas 46'
for Busquets 46'
for Sergio Ramos 66'
for Iniesta 66'
for Pedro 82'
for A. Candreva 46'
for C. Maggio 46'
for D. Criscito 46'
for Thiago Motta 62'
for A. Cerci 69'
for P. Osvaldo 69'
|Icardi pens Inter extension|
|Wambach dreaming of Women´s World Cup success|
|Blazer reveals World Cup bribes|
|FIFA sponsors will not walk away, claims expert|
|Fred relishing Copa America chance|
|Valcke protests innocence over alleged $10m bribe|
|Netherlands v United States: Klinsmann wants to see if players can compete|
|Leicester sign Fuchs on free transfer|
|Russia to continue with World Cup plans|
|Blatter´s exit must be explained, FIFPro demand|
|Iniesta pays tribute to ´teacher´ Xavi ahead of Barca bow|
|Scotland v Qatar: Hosts focused on football amid controversy|
|Lack of action may force Britton out of Swansea|
|Sunderland´s Johnson pleads not guilty to underage sex charges|
|Wilshere charged for Spurs taunts|
|Emotional Benitez vows not to change approach at Real|
|Garay wants to help Messi to Copa America glory|
|South Africa repeat denial over bribery claims|
|Interpol issues Red Notices for Warner and Leoz|
|Burnley expect Dyche stay|
|Fellaini: United will be title challengers next season|
|Premier League´s top four paid almost £100m each|
|Tuchel out to write ´new chapter´ at Dortmund|
|United wanted me to sit out PSV games - Depay|
|Platini cancels UEFA meeting following Blatter resignation|
|Lucas signs PSG extension|
|Barcelona like watching Pavarotti, says former Juve boss Capello|
|Southampton must use Koeman´s European experience - Fonte|
|Defoe highlights quartet as potential Advocaat replacements|
|Benitez takes Real reins|
|Result no concern for Herrera in Peru friendly|
|Lowy defends Australia´s bid for 2022 World Cup|
|Belgium close to major final - Pfaff|
|Chile v El Salvador: Sampaoli´s men search for form|
|Qatar hit back at Dyke|
|Monreal happy at Arsenal|
|Van Persie hints at United exit|
|Zico may run for FIFA presidency|
|Castro: Emery has Napoli offer|
|Schafer finalises Jamaica squad|
|US Soccer, FFA welcome Blatter resignation|
|Blatter decision unrelated to corruption allegations - daughter|
|CONCACAF ready to help rebuild FIFA|
|Champions League Final: Luis Enrique worried about Juve, Italian fan nears end of Berlin bike ride|
|Essien joins Panathinaikos|
|Blatter in profile: A look back on 17 years at the top of world football|
|Demichelis set for international retirement after Copa|
|Football world reacts to Blatter resignation|
|Blackpool turn to McDonald|
|Blatter resignation: How the FIFA scandal unfolded|
|Ancelotti rejects Milan approach|
|Platini: Blatter resignation ´the right decision´|
|Swiss Attorney General: Blatter not under investigation|
|Blatter resignation can inspire FIFA change - Scala|
|Mohsni faces lengthy SFA ban|
|Sepp Blatter: Resignation speech in full|
|Blatter to resign as FIFA president|
|Adkins named Sheffield United manager|
|Juve talks ongoing - Witsel|
|Costa has no intention of leaving Chelsea|
|Ligue 1: Paris Saint-Germain season review - Blanc´s men make history|
|Luis Enrique ´worried´ by Juve|
|Mourinho: Costa happy at Chelsea|
|Mourinho hits out at ´unacceptable´ Loftus-Cheek display|
|Inter an ´option´ for Richards|
|Messi: Off-field relationship with Neymar and Suarez key|
|Remy rifle seals Sydney win for Chelsea|
|Messi could replicate Bilbao goal - Luis Enrique|
|Liverpool mauling a boost in transfer market, says Stoke chairman Coates|
|Adkins: Football can learn a lot from rugby|
|Celtic swoop for City defender Boyata|
|Xavi out to sign off in style at Barca|
|Criscito and Poli leave Italy squad|
|Lacazette plays down ´unfounded´ PSG rumours|
|Lloris: No buy-out clause in Tottenham contract|
|Newcastle released Taylor and Gutierrez in same phone call|
|Alba signs new Barcelona deal|
|Benteke coy on Villa future|
|FIFA denies Valcke involvement in $10million payment|
|Brazil selection never influenced, says technical director|
|Pogba doing ´great things´ and will get better, says Deschamps|
|Bundesliga: Borussia Dortmund season review - Klopp endures rocky farewell|
|Adler ´aged three years´, Labbadia ´extremely proud´ of Hamburg|
|Colombia squad settles in Argentina|
|Balotelli ´a target´ because of Liverpool form - Richards|
|Bosnich: If I´d listened to Terry I would have avoided drugs|
|Luiz Gustavo ruled out of Brazil´s Copa America squad|
|Mexico confirm Copa squad|
|Pogba ´very happy´ at Juve|
|Pogba can be Europe´s greatest - Pirlo|
|Pinedo stays in Argentina, Bolivia captain Raldes in Copa America squad|
|Quinteros cuts Banguera, Hidalgo for Chile showpiece|
|Injured Gonzalez misses Copa America, Venezuela finalise youthful squad|
|Champions League Final: Buffon plays down Juve hopes, Iniesta doubtful|