Waving EU and Ukrainian flags, the protesters filled Kiev's iconic Independence Square and surrounding streets to bursting point to denounce Yanukovych's rejection of an EU pact under Kremlin pressure.
Significantly upping the stakes in the confrontation, demonstrators marched on the government headquarters and erected one-and-a-half metre (five feet) high barricades outside which would make it impossible for ministers to go to their offices.
Jailed ex-premier Yulia Tymoshenko said the opposition was demanding the "immediate" resignation of Yanukovych, in a no-holds-barred statement read by her daughter that was met with chants of "Resign!" from the crowds.
Soon after the speeches at the rally ended, Ukraine's Security Service (SBU) said it had opened an investigation into alleged attempts by politicians to seize power, in an apparent bid by the state to target key opposition figures.
The size of the protest, the third mass rally in successive weekends, increased the pressure on Yanukovych who further galvanised his opponents by meeting Russian President Vladimir Putin in almost total secrecy on Friday.
Police estimated the turnout at more than 60,000 and AFP correspondents said there were several hundred thousand.
In contrast to a similar rally which descended into unprecedented clashes with riot police and saw hundreds injured a week ago, the Sunday rally was peaceful and police looked on passively as protesters put up the barricades outside the government.
Putin has slammed the protests in Ukraine, saying they looked more "like a pogrom than a revolution" but the West has urged the Ukrainian authorities to heed the demands of the protest movement.
Der Spiegel reported that German Chancellor Angela Merkel and a group of European conservative parties planned to step up support for the pugilist, including through joint public appearances.
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