The Ukrainian parliament has declared President Viktor Yanukovich constitutionally unable to carry out his duties and set an early election for May 25.
Deputies in the assembly stood, applauded and sang the national anthem.
Meanwhile, Ukraine's embattled president described the country's political crisis a coup that resembled the rise of Nazis in the 1930s.
Yanukovych also said he has no intention of resigning or leaving the country.
Hours after Mr Yanukovych and opposition leaders signed an agreement aimed at resolving the country's turmoil yesterday, Mr Yanukovych went to Kharkiv in eastern Ukraine, the heartland of his support.
Today, he made the coup accusation in a televised statement.
Meanwhile, the leaders of mainly Russian-speaking regions of eastern Ukraine that are loyal to PresidentYanukovich challenged the legitimacy of the national parliament and said they were taking control of their territories.
The move appeared to increase the possibility of a split in the sprawling former Soviet republic of 46 million, despite denials by the leaders that this was their intention.
The Kiev parliament has passed a series of measures that would reduce the president's powers and pave the way to the formation of a national unity government and early presidential elections.
Mikhaylo Dobkin, Governor of Kharkiv region in northeast Ukraine, told regional leaders meeting in the city: "We're not preparing to break up the country. We want to preserve it."
But a resolution adopted at the meeting said: "The decisions taken by the Ukrainian parliament in such circumstances cause doubts about their ... legitimacy and legality."
It added: "The central state organs are paralysed. Until the constitutional order and lawfulness are restored ... we have decided to take responsibility for safeguarding the constitutional order, legality, citizens' rights and their security on our territories."
One speaker urged the creation of civilian patrols to restore order. Another said those gathered should fear reprisals if anti-Yanukovich protesters in Kiev seize power in the whole of the country.
With people at the meeting chanting "Russia! Russia!", the atmosphere contrasted with the mood in the capital Kiev where protesters want the Moscow-backed Yanukovich to resign.
Yanukovich said he had no intention of quitting or leaving Ukraine and declared all moves taken by parliament to be illegal and amounting to a "coup d'etat", Russian news agency Interfax reported, citing a television interview.
A day after Yanukovich signed an agreement with the opposition to relinquish some of his powers, his opponents were in control of the presidential administration and the Interior Ministry responsible for the police turned its back on him.
The regions represented at the meeting - Kharkiv, Donetsk, Dnipropetrovsk, Lugansk and Crimea - have a population of 14.4 million. Most are important industrial centres and Russia's Black Sea fleet is based in the Crimean port of Sevastopol.
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