Waving national flags and blowing horns and whistles, several hundred thousand foes of the embattled president packed central Caracas in the biggest anti-government demonstration in recent months.
The opposition also presented the electoral authorities with 3.2 million signatures seeking a referendum to end the controversial presidency of Chavez.
But Chavez, speaking in Argentina during an official visit, put up a brave face and said the signatures were "not valid". He said his opponents were "crazy" and "irresponsible", accusing them of using tricks and fraud to collect the signatures.
"This shows the opposition is still thinking about a coup and destabilisation", Chavez said.
But the opposition struck a buoyant note.
"The countdown to a referendum has begun," a prominent Chavez-opponent, Enrique Mendoza said.
An estimated 600,000 people are said to have participated in the march that snaked through the capital's streets for more than 1.5 km.
The opposition presented the electoral authorities with 3.2 million pro-referendum signatures.
The government, however, said the turnout was only about 50,000.
The rally and the signature campaign marked the third attempt by Chavez opponents to oust him, following a failed coup in 2002 and a general strike early this year.
The opposition needs to gather the signatures of 20% of Venezuela's 12 million registered voters to force a referendum.
Though Chavez still enjoys support from impoverished Venezuelans, he is widely loathed by the business community and large sections of the middle class, who accuse him of ruining the economy.
Chavez dismisses critics as rich "oligarchs" who fear his policies to help the poor will rob them of privileges.