Fit-again Liverpool striker Fernando Torres has been included in the Spain squad to face France on March 3.
Jesus Navas has again been included after making his debut in Spain's 2-1 victory over Argentina in November, coming on for the final eight minutes of the friendly.
The Sevilla winger has long been considered one of Spanish football's most promising players but had never played for the senior national side before, mainly due to anxiety and panic attacks when away from home for extended periods.
However, the 23-year has worked on coping with those issues and will now be hoping for more playing time as he aims to stake his claim for a place in the World Cup squad.
Alvaro Negredo has been called up again and the Sevilla striker must now be favourite to claim the final attacking place in South Africa.
Primera Division giants Barcelona and Real Madrid provide the bulk of the players, with five coming from each club.
Spain take on France at the Stade de France on Wednesday.
Squad: Casillas (Real Madrid), Reina (Liverpool), Diego Lopez (Villarreal); Sergio Ramos (Real Madrid), Pique (Barcelona), Puyol (Barcelona), Marchena (Valencia), Albiol (Real Madrid), Capdevila (Villarreal), Arbeloa (Real Madrid); Senna (Villarreal), Xabi Alonso (Real Madrid), Busquets (Barcelona), Xavi (Barcelona), Fabregas (Arsenal), Iniesta (Barcelona); Mata (Valencia), Silva (Valencia), Navas (Sevilla), Villa (Valencia), Torres (Liverpool), Guiza (Fenerbahce), Negredo (Sevilla).