Five people were injured overnight, as gunshots were fired. Rival groups blamed each other for the violence.
Earlier, Russia and the US failed to agree on how to resolve the crisis in Ukraine’s Crimea region, ahead of a secession referendum there.
Russia vowed to respect Sunday’s vote – but the US said it was illegitimate.
Moscow has been tightening its military grip on Crimea – the southern autonomous republic in Ukraine – where voters are to decide on whether to re-join Russia or stay with Kiev.
The violence reportedly began on Kharkiv’s Svoboda Square on Friday evening and later moved to an office of a pro-Ukrainian group in the city.
Eyewitnesses said that pro-Russian activists tried to storm the rival protesters, who had barricaded themselves in.
The witnesses said that shots had been fired and Molotov cocktails thrown in.
Kharkiv Mayor Hennadiy Kernes was later quoted by Ukrainian media as saying that two people were killed and five injured.
Meanwhile, Kharkiv Governor Ihor Baluta called the incident “a provocation”.
Both rival groups blame each other for starting the clashes. A criminal investigation is now under way.
This follows Thursday night’s violence in Donetsk, also in the east, where at least one person died in fighting between a pro-Russian crowd and supporters of the new government in Kiev.
Ukraine accuses Russia of using provocateurs to stoke unrest on the eastern border. Moscow denies this, vowing to protect its “compatriots” from far-right radicals.
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