Police estimated that some 18,000 people marched through the city centre, the first such demonstration by Catalan separatists in Madrid since 2012.
Many of the protesters carried the red-and-yellow striped flag of Catalonia, and a banner at the front of the march read: “Self-determination is not a crime”.
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The separatists denounce the court case mounted against the regional politicians as a politically motivated show trial.
Nine of the 12 on trial at Madrid's Supreme Court face up to 25 years in jail on charges of rebellion and other offences for pushing a secession bid referendum in October 2017 in defiance of a court ban, and for a brief declaration of independence.
The moves sparked Spain's deepest political crisis since the transition to democracy in the 1970s after the death of dictator Francisco Franco.