The Catalan chief of police, Josep Lluis Trapero, is appearing before a judge in Madrid on suspicion of sedition against the state, BBC reports.
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.