But Catalan leader Carles Puigdemont and four others have ignored the order. Prosecutors could order their arrest.
Mr Puigdemont, who is now in Belgium, said earlier: "This is a political trial."
Spain has been gripped by a constitutional crisis since the referendum was held on 1 October in defiance of a constitutional court ruling that had declared it illegal.
Last week, Spanish Prime Minister Mariano Rajoy imposed direct rule on Catalonia, dissolving the regional parliament and calling snap local elections.
This came after Catalan lawmakers voted to declare independence of the affluent north-eastern region.
The Catalan government said that of the 43% of potential voters who took part in the referendum, 90% were in favour of independence.
What is expected in the Madrid court?
On Monday, Spain's chief prosecutor said the Catalan leaders were accused of rebellion - which carries a maximum 30-year jail term - as well as sedition and misuse of funds.
They were ordered to appear in Madrid's Audiencia National (high court) on Thursday morning for questioning.
They are yet to be formally charged.
A judge will have to decide whether the officials should go to jail pending an investigation that could potentially lead to a trial.
The judge can also grant them conditional bail and order them to surrender their passports.
However, Mr Puigdemont earlier made it clear he would not be coming to Madrid.
"Those summonses are part of proceedings that lack any legal basis and only seek to punish ideas. This is a political trial," he said in a statement.
Mr Puigdemont previously said he would return to Spain if he and his colleagues received guarantees of a fair trial.
The Spanish government insists it has no influence over the country's judiciary, the BBC's Tom Burridge in Barcelona says.
But if those Catalan politicians appearing in court are denied bail it will cause further anger among those who want Catalonia to break away, our correspondent adds.
The court summons also gave them three days to pay a deposit of €6.2m ($7.2m) to cover potential liabilities.
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