Police estimated that 50,000 people jammed a plaza in front of Seoul's City Hall and an adjacent 14-lane boulevard, holding candles and anti-government signs. The protesters, including Catholic priests and Buddhist monks, sang songs and chanted slogans.
About 20,000 riot police were deployed around the site, but there were no immediate reports of clashes.
It was the second-largest rally in a series of near-daily protests held for the past two months prompted by concerns over the safety of American beef imports. A rally on June 10 drew a crowd estimated by police at about 80,000.
"Start renegotiation!" the protesters chanted, raising their candles to the sky. A large placard tied to balloons above them read "Complete renegotiation! Candles will prevail."
The demonstrators want an April agreement under which South Korea agreed to resume imports of U.S. beef rescinded and renegotiated.
The deal has sparked public outrage amid widespread perceptions, fanned in part by sensational media reports, that it exposes the country to a higher risk of mad cow disease.
The unrelenting demonstrations forced the government to negotiate an amendment to the deal with Washington last month to limit shipments to beef from younger cattle, which are believed less susceptible to mad cow disease.
The demonstrations dwindled in scale after the amendment, as violence during rallies drew criticism.
But they began drawing more people this week as religious leaders began joining them, saying they want to make sure the demonstrations are peaceful.
The government says a full renegotiation of the beef deal would hurt the country's international credibility.
The protesters are also denouncing other policies of President Lee Myung-bak, including his plan to privatize state corporations.
Lee, a conservative who took office in late February, apologized to the nation twice over his handling of the beef issue and replaced all his top advisers. He is now mulling sacking some Cabinet ministers.
Earlier Saturday, about 400 conservatives held a counter rally calling for an end to the anti-government protests.
"Stop illegal, violent candlelight demonstrations!" they shouted.
They accused their opponents of using fear of mad cow disease to undermine the conservative president.
Some demonstrators from the two opposing groups traded taunts as riot police stood between them to prevent any clashes. The pro-government demonstrators dispersed voluntarily after several hours.
South Korea banned imports of American beef in 2003, when the first of three cases of mad cow disease was discovered in the United States. Before the ban, South Korea was Nebraska's second-largest beef market, valued at $108 million annually.
U.S. beef went on sale last week for the first time under the latest import deal. But it is not widely available because large supermarket chains and most restaurants are reluctant to sell or serve it for fear of a public backlash.
Both the U.S. and South Korean governments insist U.S. beef is safe.