"The United States believes that Russia is responsible for the attack on two people in the United Kingdom, using a military-grade nerve agent," US Ambassador Nikki Haley told an emergency council meeting.
The March 4 attack in the English city of Salisbury left ex-spy Sergei Skripal and his daughter in critical condition.
US President Donald Trump had pressed Russia to provide answers but did not suggest that Moscow had a hand in the attempted murder.
Russia's ambassador repeated Moscow's denial at the council meeting and suggested that the attack was a provocation aimed at tarnishing Russia's image ahead of the World Cup and elections.
"Russia had nothing to do with this incident," said Ambassador Vassily Nebenzia. "We have nothing to fear, nothing to hide."
Haley said Russia "must account for its actions" as a permanent council member entrusted by the United Nations with upholding international peace and security.
"If we don't take immediate, concrete measures to address this now, Salisbury will not be the last place we see chemical weapons use."
Russia demanded that Britain hand over samples of the nerve agent for analysis in Moscow and suggested that if Britain was able to identify the chemical, it must also be able to produce it.
Sergei Skripal and his daughter Yulia were poisoned in March 2018. Source: AAP
Britain maintains that attack was carried out with the nerve agent Novichok, which was developed by the Soviet Union and later inherited by Russia.
"We demand that material proof be provided of the allegedly found Russian trace," said Nebenzia.
Britain called the emergency meeting to rally support in its standoff with Russia over the attack that British Deputy Ambassador Jonathan Allen said was "state-sponsored."
"We ask you today to stand by us," Allen told the council.
"This was a reckless and indiscriminate act that put at risk the lives of civilians."
UN Secretary-General Antonio Guterres earlier said the use of a nerve agent was "unacceptable" and called for a thorough investigation.
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