Jordi Sánchez, who heads the Catalan National Assembly (ANC), and Jordi Cuixart, leader of Omnium Cultural, are being held without bail while they are under investigation for sedition.
The men are seen as leading figures in organising a 1 October independence vote, which Spanish courts suspended.
The government in Madrid branded the vote illegal.
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Following the referendum, Catalonia's regional head Carles Puigdemont signed a declaration of independence, but halted its implementation to allow negotiations.
He has called for talks to take place over the next two months.
However, the Spanish government has warned that Catalonia must revoke the declaration or face direct rule from Madrid.
Mr Puigdemont has also angered Madrid by refusing to clarify whether or not he declared independence last week.
Mr Puigdemont, who has been given until Thursday to clarify his position, hit out at the government on Twitter following news of Mr Sánchez and Mr Cuixart's detention.
"Spain jails Catalonia's civil society leaders for organising peaceful demonstrations. Sadly, we have political prisoners again," he wrote.
In a video recorded before his court appearance and released on his Twitter account after his detention, Mr Cuixart instructs separatists to "never lose hope because the people of Catalonia have earned their future".
He and Mr Sánchez have also been accused of encouraging protesters as they blocked officials from entering Catalonia's regional government offices on 20 and 21 September.
Pro-independence supporters have now called for further protests, demanding the men's release, reports the BBC's James Reynolds in Barcelona.
Just hours before the ruling, the High Court freed the head of Catalonia's police force, Josep Lluis Trapero.
His force, the Mossos d'Esquadra, is accused of failing to help Spain's Guardia Civil police tackle thousands of pro-independence protesters in Barcelona during the run-up to the referendum.
Prosecutors had called for him to be held in detention
[BBC SOURCES]
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