The Catalan chief of police, Josep Lluis Trapero, is appearing before a judge in Madrid on suspicion of sedition against the state.
His Mossos d’Esquadra force is accused of failing to protect Spanish national police from protesters ahead of the 1 October independence referendum.
Another Catalan police officer and two leading independence activists are also being questioned as suspects.
The Catalan vote last Sunday was declared illegal under Spanish law.
The hearing is taking place at the national criminal court in Madrid. The defendants are accused of failing to help Guardia Civil police tackle thousands of pro-independence protesters outside the Catalan Economy Department in Barcelona on 20 September.
Leading newspaper El Pais says the accusation against the Mossos is extraordinary in post-Franco democratic Spain.
The crime of sedition has been in every Spanish penal code since 1822 and carries a potential prison term of up to 15 years. It amounts to rebellion against state decisions or national security forces.
As recently as August the Mossos was being widely praised for quickly tackling the Islamist cell that carried out the Barcelona terror attack in that month.
The Catalan regional government says it might unilaterally declare independence within days.
Spain’s Prime Minister Mariano Rajoy will chair a cabinet meeting to discuss the next moves in the confrontation with Catalonia.
Organisers of Sunday’s vote put the turnout at 42%, with 2.2 million people taking part. They say 90% voted for independence, but have not published final results. There have been several claims of irregularities.
There was violence at polling stations as police, trying to enforce a Spanish court ban on the vote, attempted to seize ballot boxes and disperse voters.
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Besides the Mossos commanders, the Madrid judge was questioning Catalan parliament president Jordi Sanchez and Omnium Cultural head Jordi Cuixart. Both are pro-independence campaigners.
The Spanish government refuses to hold negotiations on Catalonia’s independence bid. The Catalan leaders say they want international mediation and have repeatedly urged the EU to get involved.
Spain’s Constitutional Court has suspended Monday’s session of the Catalan parliament, hoping to pre-empt a possible declaration of independence.
Catalonia is Spain’s richest region and accounts for 19% of Spain’s GDP.
On Thursday, Sabadell, a major bank, decided to transfer its legally registered base from Barcelona to the south-eastern Spanish city of Alicante. Its HQ and workforce will remain in Barcelona.
CaixaBank, another large Barcelona-based institution, is reported to be considering a similar move.
This would ensure the banks remained within the eurozone and under the supervision of the European Central Bank, even if Catalonia broke away from Spain.
Catalonia in numbers
16% of Spain’s population live in Catalonia
25.6% of Spain’s exports come from Catalonia
19% of Spain’s GDP is produced in Catalonia
20.7% of foreign investment in Spain goes to Catalonia
35.3% of Catalonia’s GDP is debt
Ministry of Economy, Industry and Competitiveness, Eurostat, Bank of Spain