Some 56 people have now been released from the state security service in Luhansk, officials say
Ukraine's interior minister has warned pro-Russian activists who have taken over state buildings in eastern cities to enter talks to find a political solution or face "force".
Arsen Avakov said the situation would "be resolved in 48 hours" either way.
Earlier, some of those who had been inside security service offices in Luhansk since Sunday left the building.
The EU, Russia, US and Ukraine are to meet next week in the first four-way meeting since the crisis erupted.
The talks are aimed at breaking the impasse since Russia annexed the southern Ukrainian peninsula of Crimea in February. Russian troops are now massed along the borders of the two countries.
In another development on Wednesday, Russian President Vladimir Putin suggested state-controlled energy company Gazprom should make Ukraine pay up front for its gas, but said there should be "additional consultations" first.
Ukraine, which is on the verge of bankruptcy, owes Gazprom $2.2bn (£1.3bn) for natural gas supplies and recently missed a payment deadline.
Tensions have been high since government buildings in the eastern cities of Luhansk, Donetsk and Kharkiv were taken over by pro-Russian activists on Sunday.
Mr Avakov said an "anti-terrorist operation" was under way in the three regions and would be concluded within the next two days.
"There are two options," he told journalists, " political settlement through talks or the use of force.
"For those who want dialogue, we propose talks and a political solution. For the minority who want conflict they will get a forceful answer from the Ukrainian authorities."
The leader of the armed activists inside the state security service building in Luhansk has appealed to President Putin for help.
Calling Luhansk the "last remaining hope for all Ukraine", the man identified only as Vitaly said: "Mr Putin, have mercy on your fighters. If you lose us then you will lose the last hope to create a good neighbour."
Earlier, Ukraine's security service said 56 people held inside its Luhansk offices had been allowed to leave following two rounds of negotiations with local politicians.
During a rally outside the building overnight, speakers condemned the interim leadership in Kiev and repeated their call for a referendum on greater regional autonomy.
Ukraine's authorities said on Tuesday they had retaken control of the government building in Kharkiv.
Protesters in the regional authority building in Donetsk have been urged to leave. MP Nikolai Levchenko told reporters he feared for the safety of local residents.
"This should be resolved peacefully, he said. "I will do my best to protect them, even if they are wrong. But the most important thing is to secure the other citizens."
Barricades of barbed wire, tyres and even car bumpers surround the buildings.
A Russian flag flies by a barricade erected beside the security service building in Luhansk
These activists man the barricade outside a government building in Donetsk
Car bumpers have been used - along with tyres and barbed wire - to build barricades outside government buildings
Police officers' protective shields lie close to administrative buildings in Kharkiv
Moscow has warned Ukraine that using force to end the protests could lead to civil war.
On Wednesday, German Chancellor Angela Merkel criticised Russia for not doing enough to ease the tension.
She told parliament: "Unfortunately, in many areas it is not clear that Russia is contributing to a de-escalation of the situation."
'Genuine dialogue'
US Secretary of State John Kerry has said Russian special forces and agents had been "the catalyst behind the chaos".
President Putin says there is no intention to invade Ukraine but he reserves the right to protect Russian interests there.
Russian Foreign Minister Sergei Lavrov will meet Mr Kerry, EU foreign policy chief Catherine Ashton and Ukraine's Foreign Minister Andriy Deshchytsia next week.
Senior US diplomat Victoria Nuland said expectations were not high for the talks but it was important to keep the "diplomatic door open".
Mr Lavrov, who spoke to Mr Kerry by phone on Wednesday, said the talks should focus on "dialogue among Ukrainians" and not on bilateral relations among the participants.
Kiev and its allies accuse Moscow of fomenting unrest in the mainly Russian-speaking east of the country as a pretext to possibly seizing more territory - a claim strongly refuted by Russia.
On Wednesday, Moscow again denied any intent behind its forces being stationed along the Ukraine border.
Moscow has so far refused to recognise the new authorities in Kiev following the ousting of pro-Russian President Viktor Yanukovych in February.
Crisis timeline
- 21 Nov 2013: President Viktor Yanukovych abandons an EU deal
- Dec: Pro-EU protesters occupy Kiev city hall and Independence Square
- 20-21 Feb 2014: At least 88 people killed in Kiev clashes
- 22 Feb: Mr Yanukovych flees; parliament removes him and calls election
- 27-28 Feb: Pro-Russian gunmen seize key buildings in Crimea.
- 16 Mar: Crimea voters choose to secede in disputed referendum
- 18 Mar: Russian and Crimean leaders sign deal in Moscow to join the region to Russia
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