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'
|Burton and Day depart West Brom|
|Mendilibar makes Eibar return|
|Milan agree to terminate Muntari contract|
|Copa final a dream come true for Sampaoli|
|CSKA Sofia demoted due to financial crisis|
|Wolfsburg warn off De Bruyne suitors|
|Kone: I want to prove I can cut it at Everton|
|West Ham v Lusitans: Bilic set for first game in charge|
|Millennium Stadium to host 2017 UEFA Champions League final|
|Ronaldo a ´possibility´ for Portugal´s Olympics squad|
|Liverpool should get rid of Sterling - Grobbelaar|
|Miranda arrives for Inter medical|
|Arsenal can challenge the best - Cech|
|Real star Bale can be better than Ronaldo, says Keegan|
|UEFA approves changes to FFP rules|
|Honduras v Mexico: Moreno´s injury may force Mexican rethink pre-Gold Cup|
|Hiddink was the wrong man for Netherlands - De Boer|
|Vargas: I was scoring with every shot at training|
|Zambrano red rocked Peru - Gareca|
|Paraguay to change tack against Argentina|
|Sampaoli thrilled with substitutes´ influence, applauds Peru|
|Martino rues Copa America date with former side Paraguay|
|Former Birmingham City player Thwaites among Tunisia victims|
|Chile 2 Peru 1: Zambrano´s red card helps hosts into Copa final|
|Thiago Silva: ´I didn´t even know the ball had hit my hand´|
|Ronaldinho set to sign for Antalyaspor|
|Jara ban reduced on appeal by one match|
|Hiddink leaves Netherlands post|
|Cresswell pens new five-year deal at West Ham|
|Koeman: No serious United bid for Schneiderlin|
|Barbados eliminated from World Cup qualifying after fielding ineligible player|
|Former Catania president admits buying matches|
|Jones agrees Manchester United deal|
|Argentina v Paraguay: Valdez hoping not to need another comeback|
|Milan seal Bertolacci signing|
|Cech grateful to Abramovich and Chelsea fans|
|UEFA Champions League Preview: Brotherly pride at stake as B36 play TNS|
|UEFA Europa League Preview: Tommasi set to aid La Fiorita qualifying bid|
|Cech leaves Chelsea to join Arsenal|
|Paulinho ends Spurs frustration with Guangzhou move|
|Sampdoria confirm Eto´o exit amid Antalyaspor talk|
|Garcia departs Espanyol ahead of expected Qatar move|
|Watford to delay Doyley decision|
|Schlupp agrees new Leicester deal|
|Schweinsteiger hints at Bayern stay amid United links|
|Bertolacci thrilled ahead of Milan move|
|Langerak leaves Dortmund for Stuttgart|
|Muniain extends Athletic contract|
|Youngsters to get chance on US tour - Van Gaal|
|La Liga chief hits out at Real Madrid over TV rights opposition|
|Villa confirm Wilkins as assistant manager|
|Dicks joins West Ham coaching staff|
|Heldt enters to jeers, exits to cheers|
|Paraguay pride has grown at Copa America - Aguilar|
|Van Gaal tight-lipped on De Gea|
|Bacca confirms Milan agreement|
|Weight of expectation on hosts Chile to benefit Peru - Gareca|
|MLS Review: Timbers thump Sounders thanks to Adi´s double|
|Dzeko ´would run to Roma´ - Pjanic|
|Peru won´t sit back - Sampaoli|
|Zenga: Cassano, Balotelli will not be joining Sampdoria|
|Victory to start A-League defence away to Adelaide|
|I want to play MLS ´at the peak of my career´ - Johannsson|
|Allegri: I like Isco and Oscar|
|CAF Champions League Review: Comeback kings Smouha go top of Group A|
|Pirlo in attendance for New York derby|
|No Bayern deal yet, states Costa|
|Jara handed three-game ban|
|Sunderland striker Defoe sets 20-goal target|
|Leicester offer condolences to Albrighton and family after Tunisia attack|
|Chile v Peru: Potential Jara sanction hangs over semi-final|
|Sweden U21 v Portugal U21: History beckons for Jorge´s men|
|Neville appointed Valencia number two|
|Work halted at Olympic Stadium after fatality|
|Ibrahimovic in the same league as Messi - Sacchi|
|Giggs: United want to add winners|
|Turan sets record straight over transfer talk|
|Low keen for new Germany leaders to emerge|
|Tunisia part company with Leekens|
|Evans: Sterling´s Anfield exit must be a done deal|
|FIFA audit chief urges Blatter to stick by decision|
|Hrubesch urges caution after Germany U21s humbled|
|Taarabt hits out at ´very bad manager´ Redknapp|
|Bruce: I´m lucky I wasn´t sacked after Hull relegation|
|Van Hooijdonk warns Van Gaal over Depay tactics|
|SPFL looking into winter break|
|MLS Review: Earthquakes win California Clasico, Vancouver claim top spot in West|
|Muto must toughen up to succeed at Mainz - Ficcadenti|
|Diaz: Paraguay refuse to lose|
|Tragedy strikes for Paraguay match-winner Gonzalez|
|Robinho rues substitution, wanted to take penalty|
|Dunga: Brazil squad hit by virus|
|Fiorentina make ´insane offer´ to Salah|
|Women´s World Cup Review: England reach maiden semi-final|
|Mexico 2 Costa Rica 2: Herrera´s men recover after half-time|
|ES Setif 1 USM Alger 2: African champions beaten|
|Paraguay 1 Brazil 1 (4-3 on penalties): History repeats as Dunga´s men bow out|