Players from Real Zaragoza and Levante have been issued with a summons as part of an investigation into match-fixing.
The game under investigation took place at the end of the 2010-11 season, when Zaragoza won 2-1 at Levante.
Current Atletico Madrid captain Gabi Fernandez scored twice for Zaragoza in a victory that saved the club from relegation.
Both clubs deny any wrongdoing and say they will co-operate fully with the enquiry.
The state prosecutor's office in Madrid said in a statement that it had "started procedures to investigate match-fixing and has summoned players implicated for questioning on 2 October".
In June 2013, Spanish league president Javier Tebas included the Levante-Zaragoza match in a list of nine matches that the league was investigating for possible match-fixing.
If the state prosecutor finds evidence of a crime, the case will be brought to a judge. Match-fixing is a crime in Spain and can lead to prison sentences for individuals and a club being banned from competition.
Uefa president Michel Platini recently spoke about how match-fixing is the biggest threat to the future of football.