The huge demonstration on Saturday was the largest by the opposition this year and followed a week of violent clashes between troops and protesters in which at least eight people died.
Venezuelan opposition leaders said the big turnout gave fresh impetus to their campaign for Chavez to submit to a vote and resolve the political conflict that has embroiled the world's fifth largest oil exporter for more than two years.
"We don't want bullets, we want votes," said Eduardo Fernandez, a veteran opposition Christian-Democrat politician.
But the National Electoral Council has so far only validated 1.8 million signatures of a petition that could trigger a referendum on leadership - short of the 2.4 million required by law.
Arguing over signatures
Opposition leaders are contesting the decision by the electoral council on Tuesday requiring that one million pro-referendum signatures be reconfirmed.
"The only way to solve this is through a vote," said university professor Roberto Rodriguez, a pro-referendum marcher.
"Otherwise we're going to be killing each other," another protester, Nicolas Gonzales added.
The United States, the biggest buyer of Venezuelan oil, rejects Chavez's allegations of supporting the opposition as an attempt to deflect attention from his domestic problems.
But speaking from his Texas ranch, US President George Bush said Washington would work with the Organisation of American States "to help ensure the integrity" of the referendum process.