Explosions are taking place, fireworks are being thrown and large fires have broken out in Independence Square.
On Tuesday at least 18 people were killed, including seven policemen, in the worst violence seen in weeks.
President Viktor Yanukovych blamed the violence on opposition leaders, but said it was still "not too late to stop the conflict".
He was speaking after a late-night meeting with opposition figures Vitaly Klitschko and Arseniy Yatsenyuk.
Mr Klitschko, who leads the Udar (Punch) party, told Ukraine's Hromadske TV that the president had given the protesters only one option, leave the Maidan and go home.
'Island of freedom'
Security forces had given protesters a deadline of 18:00 local time (16:00 GMT) to leave Independence Square, the scene of a mostly peaceful protest camp since November.
The city's metro service was completely shut down, and there were reports that cars were being prevented from coming in to the capital.
Then shortly before 18:00 GMT, police announced over loudspeakers that they were about to begin "an anti-terror operation".
They advanced with an armoured vehicle, dismantling barricades and firing stun grenades and water cannon.
Protesters threw fireworks and petrol bombs, and lit fires to block off police. Many tents have been burned but it was unclear whether there had been casualties.
Large fires are burning in the square, including around these statues of Kiev's founders
Protesters have remained in the square, some setting off fireworks
Protesters have insisted they will not leave the Maidan
Late on Tuesday, the police tried to break through a barricade from the Evropeyska Square, but the attack was repelled.
In a renewed assault shortly after 04:00 local time on Wednesday (02:00 GMT), the police tried to move on the protesters' tents near the main monument on the square.
A number of tents were set ablaze, and the police reportedly again began using water cannon.
In speeches from the main stage, protest leaders urged people already on the Maidan to stand firm, and called on Ukrainians elsewhere to come to the square.
"This is an island of freedom and we will defend it," said Mr Klitschko.
Mr Yatsenyuk, who heads the Fatherland party, appealed to President Yanukovych to "stop the bloodshed and call a truce".
Former deputy prime minister and activist Oleg Rybachuk: "The people are surrounded"
Meanwhile, there are reports of unrest breaking out elsewhere in Ukraine, including the western cities of Lviv, Ivano-Frankivsk.
Debate 'blocked'
The BBC's David Stern in Kiev says this is a key moment for the country and that many people are scared of further escalation. Although this does not necessarily mean a civil war - as some have previously suggested - Ukraine remains dangerously divided, our correspondent says.
Ukraine's unrest began in November, when President Yanukovych rejected a trade deal with the EU in favour of closer ties with Russia.
Protesters were earlier blocked by police as they tried to march on parliament
Pro-EU protesters demanded his resignation and snap elections.
After weeks of unrest, the mood had calmed in recent days, but people remained on the streets.
Earlier on Tuesday, police blocked protesters from marching on parliament, where MPs had been due to debate proposed changes to the constitution which would have reduced the powers of the president.
The debate did not take place. Mr Yatsenyuk said President Yanukovych was blocking the reforms and that his allies "show no desire whatsoever to end the political crisis".
But MPs who support the president said the proposals had not been thoroughly discussed, and that more time was needed.
Some protesters outside parliament ripped up cobblestones to throw at police. Police fired stun and smoke grenades, and rubber bullets. Correspondents say it was unclear what sparked the latest violence, with each side blaming the other.
Protesters also attacked the headquarters of President Yanukovych's Party of the Regions, temporarily smashing their way in and setting it on fire before being forced out by police.
Police said late on Tuesday that at least 16 people had been killed, including seven police officers.
The White House said it was "appalled" by the violence, saying "force will not resolve the crisis".
Spokesman Jay Carney urged President Yanukovych to "restart a dialogue with opposition leaders today".
White House Press Secretary Jay Carney: "Force will not resolve the crisis"
UN Secretary General Ban Ki-moon had earlier called for restraint and dialogue.
In other international messages of concern:
German Foreign Minister Frank-Walter Steinmeier said Ukraine was "experiencing dramatic hours" and that the EU could consider imposing sanctions.
The UK's Minister for Europe, David Lidington, said such violence had "no place in a European democracy"
EU foreign policy chief Catherine Ashton earlier said she was "deeply worried" about the unrest and urged politicians to "address the root causes"
Russia blamed the earlier violence on "connivance by Western politicians" and their refusal to consider the "aggressive actions" of radical factions within the protest movement
Rinat Akhmetov, one of Ukraine's richest men - and a powerful financial backer of Mr Yanukovych - said there were "no circumstances that would justify the use of force against peaceful citizens".
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