Chants of "revolution'' had earlier resounded across a sea of EU and Ukrainian flags on the square, where the government had banned rallies starting on Sunday.
But far-right nationalist leader Oleh Tyahniboh called for a national strike, and members of his Svoboda (Freedom) party occupied Kiev's city hall along with followers of former economy minister Arseny Yatsenuk's Batkivshchyna (Fatherland) Party.
All three opposition leaders also occupied a trade union building, turning it into a temporary headquarters.
The events, evoking memories of the 2004-5 Orange Revolution that overturned the established political order, took place against the background of an apparent attempt by a protesters to storm the main presidential office.
Interior Ministry forces and riot police kept the protesters, who used an earth excavator in an attempt to break through police lines, at bay.
Police said 100 officers had been injured in violence during the day, news agencies reported.
'They stole the dream'
But opposition politicians, who had been urging protesters all day to remain peaceful, denounced the violence at the headquarters of President Viktor Yanukovich's administration as a stage-managed "provocation" to justify a security clampdown.
They sent officials to appeal to supporters to return to the main protest on Independence Square.
Opposition leader Vitaly Klitschko, addressing the protesters, called on Yanukovich and his government to resign, saying they had "stolen" Ukraine's dream of European integration.
"The government and the president should resign," said the heavyweight boxing champion turned opposition politician.
"They stole the dream. If this government does not want to fulfill the will of the people, then there will be no such government, there will be no such president. There will be a new government and a new president," he said to cheering crowds.
'We are furious'
The opposition rally came a day after a police crackdown on protesters which inflamed demonstrators further after Yanukovich's U-turn on Europe.
In a bid to defuse tensions ahead of the rally, Yanukovich issued a statement saying he would do everything in his power to speed up Ukraine's moves toward the European Union.
In a sea of blue and gold, the colours of both the EU and Ukrainian flags, protesters flooded the streets of central Kiev, angered by Yanukovich's decision last month to forego signing a landmark EU deal in favour of closer ties with Kiev's former Soviet master, Russia.
"We are furious,'' said Mykola Sapronov, a 62-year-old retired businessman.
"The leaders must resign. We want Europe and freedom.''
Sunday's rally also marked the anniversary of a 1991 referendum that ushered in Ukraine's independence from the then-crumbling Soviet Union.
Source: Al Jazeera