Spain coach Julen Lopetegui has left Cesc Fabregas and Cesar Azpilicueta out of his squad for next month's World Cup qualifiers against Italy and Albania.
Fabregas, who has struggled to make himself a regular fixture for Chelsea so far this season, was not included in Lopetegui's maiden squad in August.
His Chelsea team-mate Azpilicueta, 27, was named in the side but has been dropped for Spain's latest round of fixtures.
Elsewhere in-form Napoli star Jose Callejon has been called up to represent his country.
Callejon has not featured for La Rioja since a pair of substitute appearances in 2014 but has been in superb form this season, with five goals in six Serie A appearances.
Real Madrid defender Nacho and Sevilla goalkeeper Sergio Rico are also in having missed out on Lopetegui's first squad for the September internationals against Belgium and Liechtenstein, while Barcelona captain Andres Iniesta is back from injury.
Isco returns to the national team fold despite playing just 54 La Liga minutes for Madrid this season.
Marco Asensio, who has been more prominently involved with Madrid than Isco this term, will drop down to the Under-21 squad while injured Borussia Dortmund centre-back Marc Bartra has not made the cut along with Barcelona forward Paco Alcacer.
"Callejon can give us a lot of things," Lopetegui said. "He is at a very high level.
"Sergio Rico has also been playing well. We want to have him here.
"Fabregas has played a little bit more at Chelsea. We like Cesc, but we have to choose. There are a lot of players and we can't call them all. I hope he can play more minutes.
"Isco is a player I like. I believe in him. I would like him to play more minutes at Real Madrid.
"Marco Asensio has a present and a future, but we evaluated other options for these two matches."
Spain squad in full:
Goalkeepers: Pepe Reina (Napoli), David De Gea (Manchester United), Sergio Rico (Sevilla)
Defenders: Nacho (Real Madrid), Jordi Alba (Barcelona), Dani Carvajal (Real Madrid), Sergio Ramos (Real Madrid), Gerard Pique (Barcelona), Javi Martinez (Bayern Munich), Sergi Roberto (Barcelona)
Midfielders: Koke (Atletico Madrid), Saul Níguez (Atletico Madrid), David Silva (Manchester City), Sergio Busquets (Barcelona), Thiago Alcantara (Bayern Munich), Andres Iniesta (Barcelona), Isco (Real Madrid)
Forward: Nolito (Manchester City), Jose Callejon (Napoli), Lucas Vazquez (Real Madrid), Vitolo (Sevilla), Diego Costa (Chelsea) and Alvaro Morata (Real Madrid)