The 2018 World Cup qualification encounter between Spain and Italy will be played at the Estadio Santiago Bernabeu on September 2.
The capital has been chosen by the Spanish Football Association as the venue for what will be a key fixture in La Roja's bid to qualify for Russia.
Spain have not played a competitive fixture at the home of Real Madrid since March 28, 2009 when they faced Turkey in a World Cup qualifier, but they have played there once since - when the then reigning world champions hosted Colombia in a friendly in 2011.
The game is also set to hold special significance for Italy, as they return to the venue where they won the 1982 World Cup final.