The protest comes three weeks after a popular uprising toppled pro-Russian president Viktor Yanukovych, which was followed by the occupation of Ukraine's Crimea Peninsula by forces loyal to Moscow.
It illustrates the fine line that the new authorities in Kiev have to tread in the mainly Russian-speaking east on the eve of a referendum in Crimea on joining Russia.
"Referendum, Referendum," the crowd of at least 5,000 chanted in Lenin Square before marching on the headquarters of the SBU intelligence agency in Donetsk.
The crowd was demanding the release of Pavel Gubarev, the self-declared "people's governor" of the Donetsk region, whom the Ukrainian authorities arrested on March 6 and are investigating for separatism.
"The people are angry with the police and government, who are clamping down on our protesters," said Robert Donia, who describes himself as Gubarev's deputy.
"They freed the Maidan protesters (pro-Europeans who ousted Yanukovych) but have kept ours in jails. Why do their laws work only for them?" he told AFP before leading a rally cry of "Referendum, Referendum!"
But when this passed unanswered, tempers flared and some furious protesters barged through the front door and smashed glass panels of the SBU building.
Some demonstrators at the back of the crowd urged them to stop, before Donia appeared and declared that he had negotiated an agreement for Gubarev's release tomorrow.
"If they don't free him tomorrow, we'll be back with twice as many people," he told the crowd.
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