The accusation came as a team of UN inspectors was in Syria to probe previous allegations of chemical weapons strikes levelled against both sides during the 29-month conflict.
Western governments demanded an immediate investigation into the new allegations. Russia, a longstanding ally of the Damascus regime, echoed the call but said it suspected a "provocation" by the opposition and its foreign backers.
Videos distributed by activists, the authenticity of which could not immediately be verified, showed medics attending to suffocating children and hospitals being overwhelmed.
People inspecting bodies of children and adults laying on the ground as Syrian rebels claim they were killed in a toxic gas attack by pro-government forces in eastern Ghouta (AFP)
More footage showed dozens of people laid out on the ground, among them many children, some of them covered in white sheets.
The claim of chemical weapons use, which could not be independently confirmed, was vehemently denied by the Syrian regime which said it was intended to hinder the work of the UN weapons inspectors already in the country.
Opposition sources accused the army of multiple chemical weapons strikes -- one in Moadamiyet al-Sham, southwest of Damascus, and more in the capital's eastern suburbs.
The Local Coordination Committees (LCC), a network of activists, reported hundreds of casualties in the "brutal use of toxic gas by the criminal regime".
And in videos posted on YouTube, the Syrian Revolution General Commission, another activist group, showed what it called "a terrible massacre committed by regime forces with toxic gas."
The attack "led to suffocation of the children and overcrowding field hospitals with hundreds of casualties amid extreme shortage of medical supplies to rescue the victims, particularly atropine," the LCC said.
In one video, children are seen being given first aid in a field hospital, notably oxygen to help them breathe. Doctors appear to be trying to resuscitate unconscious children.
Specialists in the impact of chemical weapons said the video evidence was not entirely convincing.
Bodies of children laying on the ground as Syrian rebels claim they were killed in a toxic gas attack by pro-government forces in eastern Ghouta (AFP)
"At the moment, I am not totally convinced because the people that are helping them are without any protective clothing and without any respirators," said Paula Vanninen, director of Verifin, the Finnish Institute for Verification of the Chemical Weapons Convention.
"In a real case, they would also be contaminated and would also be having symptoms."
John Hart, head of the Chemical and Biological Security Project at Stockholm International Peace Research Institute said he had not seen the telltale evidence in the eyes of the victims that would be compelling evidence of chemical weapons use.
"Of the videos that I've seen for the last few hours, none of them show pinpoint pupils... this would indicate exposure to organophosphorus nerve agents," he said.
Gwyn Winfield, editor of CBRNe World magazine, which specialises in chemical weapons issues, said the evidence did not suggest that the chemicals used were of the weapons-grade that the Syrian army possesses in its stockpiles.
"We're not seeing reports that doctors and nurses... are becoming fatalities, so that would suggest that the toxicity of it isn't what we would consider military sarin. It may well be that it is a lower-grade," Winfield told AFP.
The opposition National Coalition's George Sabra, who spoke to reporters in Istanbul, said more than 1,300 people had been killed in what he said was a "coup de grace that kills all hopes for a political solution in Syria".
"The Syrian regime is mocking the UN and the great powers when it strikes targets near Damascus, while the (UN weapons inspectors) are just a few steps away," he said.
State news agency SANA said "reports on the use of chemical weapons in Ghouta (the Damascus suburbs) are totally false. It's an attempt to prevent the UN commission of inquiry from carrying out its mission."
The news agency described Wednesday's violence as "a series of operations" by army units "against terrorist groups" in Jobar, Irbin and Zamalka, "killing a number of them and destroying their hideouts".
In a statement, the army flatly denied as "null, void and totally unfounded" the opposition's allegations, describing them as a "desperate bid to conceal their failures on the battlefield".
Britain, France and the United States sought an urgent meeting of the UN Security Council to discuss the allegations.
Washington has previously described chemical weapons use as a red line that might prompt it to intervene militarily in Syria.
EU foreign affairs chief Catherine Ashton said the allegations "should be immediately and thoroughly investigated."
The UN mission already in Syria "must be allowed full and unhindered access to all sites," she said.
Russia, which has previously said it has proof of chemical weapons use by the rebels, expressed deep scepticism about the opposition's claims.
The foreign ministry said the timing of the allegations as UN inspectors began their work "makes us think that we are once again dealing with a premeditated provocation."
There have frequent claims by anti-regime activists of chemical weapons use by the army, particularly in Damascus province and in the central region of Homs.