Venezuela police are out in force in Caracas
Demonstrators have marched on the capital from six different directions.
Protests have erupted in the last week over the electoral council's refusal to authenticate hundreds of thousands of signatures collected by the opposition.
Extra police were deployed ahead of Saturday's march and civilians have been banned from carrying firearms.
At least eight people have been killed in anti-government protests in recent days.
Dressed in the yellow, blue and red colours of Venezuela's flag, protesters blew whistles and chanted anti-Chavez slogans as they marched towards the centre of Caracas.
"We're prepared to take to the streets a thousand times until we're allowed the recall referendum," opposition leader Henry Ramos Allup said.
"Nobody is going to rob us of our right to oust Hugo Chavez peacefully," he added.
At least 2.4 million signatures are required to trigger a referendum on the president. The opposition says it has collected 3.2 million.
But the electoral council ruled last week that only 1.8 million were valid.
It ordered 1 million people to confirm that they completed the petition form, because they were said to have violated procedural rules.
Another 140,000 signatures were rejected outright.