The council in Donetsk region in the Donbass coalfield voted on Sunday 156-1 to stage the vote in response to efforts to overturn Yanukovich's victory in last week's presidential election.
"We see no other way to defend the interests of Donbass," said council head Boris Kolesnikov.
In his reaction, liberal presidential candidate Viktor Yushchenko warned that officials in eastern regions promoting autonomy pose a threat to Ukraine and will be held criminally responsible for their actions.
Mass protests called
A Ukrainian opposition reacted to the referendum by urging tens of thousands of demonstrators in Kiev to give outgoing President Leonid Kuchma "24 hours" to dismiss pro-Russia Prime Minister Viktor Yanukovich.
Protesters have taken to the
streets in sub-zero temperatures
"We are giving Leonid Kuchma a deadline. He must fire Viktor Yanukovich, the separatist governors of Donetsk, Luhansk and Kharkiv and start legal action," opposition lawmaker Yulia Timoshenko said.
"We give him 24 hours to do that," she added. "If not, we'll consider him a criminal and start legal action."
Timoshenko also asked the army "to defend the [Ukrainian] people".
"I appeal to the army. Do not leave the people defenceless," she said, after telling the crowd that the authorities were planning to crack down on demonstrators overnight.
Addressing supporters in the Ukrainian capital Kiev on Sunday, Yushchenko said officials in the eastern part of the country "could take a decision which would threaten the country's territorial integrity".
"Those people who will raise the issue of separatism will be held criminally responsible under ... the Ukrainian constitution," he said. Eastern Ukraine voted solidly for Yanukovich.
Yushchenko demanded that prosecutors open a criminal inquiry against regional leaders threatening to declare autonomy. "We demand the opening of a criminal inquiry against the separatist governors."
Ukraine's parliament had earlier boosted opposition supporters by declaring invalid last week's presidential poll that handed victory to Yanukovich.
Prime Minister Victor Yanukovich
bagged the pro-Russian vote
However, Saturday's ruling, which highlighted widespread fraud in the elections, has no legal force as parliament claims no jurisdiction over the validity of elections.
The declaration was issued two days before a supreme court case examining Yushchenko's complaints of poll irregularities and as the European Union said it wanted to see new elections.
The overwhelming vote, which also expressed no confidence in the central election commission overseeing the election, was taken a day after the rivals agreed at a meeting with mediators to meet regularly to resolve their differences.
"This was an extraordinarily difficult day," parliament speaker Volodymyr Lytvyn told Interfax Ukraine news agency. "Political leaders will now have to objectively analyse the possibilities and limits of compromise."
That prospect looked increasingly distant on Sunday, with the outgoing President Kuchma saying the talks between the feuding rivals for the presidency were going badly.
In a televised statement, Kuchma also said that Yushchenko was not showing "goodwill" in lifting a blockade by his supporters of government offices.
Earlier, Yanukovich, and Moscow mayor Yury Luzhkov attended a meeting of Ukraine's Russian-speaking regions where local officials demanded autonomy.
The pro-Russian Yanukovich attended the meeting of 3500 local officials from 17 regions in the eastern city of Severodonetsk where threats of declaring autonomy were made, Interfax news agency reported.
"We call on all cities to hold votes of no confidence in the government and the creation of a new Ukrainian government in the form of a federal republic"
Boris Kolesnikov, eastern Donetsk region council head
As the political winds appear to shift in favour of Yushchenko, eastern and southern Russian-speaking regions of Ukraine have stepped up autonomy demands.
Yushchenko is supported by the liberal-oriented western half of the country.
"We had hoped for a peaceful resolution of the crisis, but Saturday's parliamentary meeting showed it wasn't to be," Donetsk council leader Kolesnikov was quoted as saying by Itar-Tass news agency.
"Therefore we call on all cities to hold votes of no confidence in the government and the creation of a new Ukrainian government in the form of a federal republic, the most democratic form of government," he said.