The opposition Syrian National Coalition has denied a Russian claim that rebel fighters fired a projectile laden with the nerve agent sarin at a suburb of Aleppo in March.
It said UN inspectors should be allowed to investigate the attack.
Separately, western diplomats said Russia blocked a draft UN Security Council resolution this week calling for a stalled UN chemical weapons investigation team to be allowed to visit Syria and to be permitted to conduct an "objective" inquiry.
Russia, along with Iran, is Syria's closest ally and chief arms supplier.
The draft resolution echoed a recent statement by the Group of Eight (G8) developed nations including Russia.
"The Free Syrian Army strongly condemns all usage of chemical weapons against a civilian population and denies Russia's allegations about the FSA using chemical weapons in Khan al-Assal, Aleppo," Khalid Saleh, a spokesman for the coalition, said in a statement.
"Only the Assad regime has the know-how, capability and willingness to use these weapons," Mr Saleh said, referring to Syrian President Bashar al-Assad.
"The coalition and supreme military council have asked for the UN monitors to come to Syria to investigate the use of these weapons and the Assad regime refuses to allow them to do so," he said.
Russia's UN envoy Vitaly Churkin last night said Russian scientific analysis strongly indicated a projectile containing sarin that hit Khan al-Assal on 19 March, killing 26 civilians and military personnel, was fired by rebels.
The government and rebels have blamed each other for that incident, as well numerous other alleged chemical attacks.
Both sides deny using chemical weapons themselves.
"The usage of chemical weapons is inconsistent with the guiding principles and goals of the Syrian revolution," Mr Saleh said.
"Targeting civilians indiscriminately to achieve political gains is a common characteristic of the Assad regime."
The United States has cast doubt on the Russian analysis of the Khan al-Assal incident and, along with France, called for full UN access to Syrian sites where chemical weapons use was suspected.
The United Nations says as many as 100,000 have died in the two-year civil war.
Speaking on condition of anonymity, a senior western diplomat also expressed scepticism about the Russian claim that the rebels were behind the Khan al-Assal attack.
He dismissed the idea that Assad's government was willing to let the UN team investigate Aleppo because it was certain the rebels were responsible for the 19 March chemical attack.
He said available evidence suggested the Syrian army carried out the attack.
"What they hope will be discovered there is lots of soldiers who were poisoned by chemical weapons, which is true," the envoy said.
"But our information suggests that that was because the projectile ... fell short and landed in an area where there were Syrian troops, not that the opposition had done it."
Mr Churkin said Russian experts visited the location where the projectile struck and took their own samples of material from the site.
Those samples, he said, were then analysed at a Russian laboratory certified by the Organization for the Prohibition of Chemical Weapons.
He also said that the projectile was not a standard military weapon.
Speaking on condition of anonymity, western diplomats on the 15-nation Security Council said Russia blocked a draft resolution based on a statement Moscow supported at last month's G8 summit in Belfast that urged all parties to the conflict to grant access to the UN team "in order to conduct an objective investigation into reports of the use of chemical weapons".
Diplomats said Russia justified its opposition to the resolution by saying the timing was not right.
Russia's UN mission did not respond to a request for comment.
So far, chief UN chemical weapons inspector Ake Sellstrom's team has not travelled to Syria because of diplomatic disputes over the scope of access he would have there.
UN Secretary-General Ban Ki-moon wants Mr Sellstrom to have unfettered access to investigate all credible alleged chemical attacks while Assad's government wants the UN experts to confine their investigation to the 19 March incident.
That disagreement has caused a deadlock in talks between the United Nations and Syria on access for the inspection team.
Syrian UN Ambassador Bashar Ja'afari on Monday said his government has invited Mr Sellstrom and UN disarmament chief Angela Kane to Damascus to discuss allegations of banned arms use in Syria's two-year civil war but suggested it would not compromise on access.
The senior Western diplomat said Mr Sellstrom and Ms Kane were expected to accept the invitation and travel to Damascus soon to discuss ways of breaking the deadlock.