If the four lawmakers refuse to cede their seats then the move could reduce the threshold of an absolute majority that acting Prime Minister Pedro Sanchez needs in the congress, or lower house, to be sworn in for a second term.
However, if the four hand over their parliamentary mandates to like-minded replacements, Sanchez would still need the help of Catalan separatist parties to be sworn in during an investiture vote expected in the first week of July. His Socialists came top in a general election last month but failed to secure a majority in the assembly.
The mandates of Oriol Junqueras, Jordi Turull, Josep Rull and Jordi Sanchez were automatically suspended because they are being held in custody while on trial in the Supreme Court, parliament speaker Meritxell Batet told a news conference. She said she had asked for a report to clarify what the threshold for an absolute majority will be in this case.
The four men have been charged with rebellion for organising a banned independence referendum which was followed by a short-lived declaration of independence, sparking Spain's worst political crisis in decades. The first three have also been charged with embezzlement of public money to stage the referendum, which was marred by a violent police crackdown.
Catalan senator Raul Romeva, who is also on trial for rebellion and embezzlement, was expected to be suspended later by the Senate, parliament's upper house.
The Supreme Court allowed the five Catalan leaders to temporarily leave jail on Tuesday to be sworn in along with other newly-elected lawmakers. They are among 12 Catalan leaders to go on trial since February, with a
ruling in the highly sensitive proceedings expected during the second half of the year.
Junqueras, a former Catalan vice-president who heads the separatist ERC party, is the lead candidate in Sunday's European Parliament election for a European regionalist party that defends the right to self-determination, the European Free Alliance.
His rival, former Catalan president Carles Puigdemont who fled to Belgium after the failed independence declaration, will also stand in the European elections as the leading candidate for his Junts per Catalunya (JxCat), the other main separatist party.
The two hope to use their positions in the European Parliament to denounce what they consider to be Madrid's heavy-handed repression.
“We have been suspended in Spain but they can't do it in Europe!,” Junqueras tweeted. “A political prisoner entering the European Parliament is the best way to denounce the repression of the Spanish State.”
Conservative parties, including upstart far-right party Vox which won seats in parliament for the first time last month, accuse Sanchez and his left-wing allies of preparing to make concessions to the separatists in exchange for support in parliament.
Sanchez argues he is only seeking dialogue with the separatists in the hope of defusing the Catalan crisis.
A poll published on May 10 by the Catalan government's CEO survey institute showed that slightly more Catalans were against independence than were in favour — the first time that has been the case since June 2017.
By AFP's Patrick Rahir