"We have indications of the use of a toxic industrial chemical" in the town of Kfar Zeita, State Department spokeswoman Jen Psaki said, referring to a rebel-held area.
"We are examining allegations that the government was responsible," she told a regular news briefing. "Obviously there needs to be an investigation of what's happened here."
Syrian opposition activists reported that helicopters dropped chlorine gas on Kfar Zeita on April 11 and 12. The U.S. ambassador to the United Nations, Samantha Power, told ABC's "This Week" on April 13 that the attack was "unsubstantiated."
Psaki said chlorine was not one of the priority one or two chemicals Syria declared to the Organization for the Prohibition of Chemical Weapons under a Russian-U.S. agreement for the destruction of Syria's chemical weapons stockpile.
A U.N. inquiry found in December that sarin gas had likely been used in Jobar, on the outskirts of Damascus, in August and in several other locations, including in the rebel-held Damascus suburb of Ghouta, where hundreds of people were killed.
The Ghouta attack caused global outrage and a U.S. threat of military strikes that was dropped after President Bashar al-Assad pledged to destroy his chemical weapons arsenal.
The Syrian government failed to meet a Feb. 5 deadline to move all of its declared chemical substances and precursors, some 1,300 tonnes, out of the country. It has since agreed to remove the weapons by late April.
Rebel activists posted photographs and video they said showed an improvised chlorine bomb to back up their claims about Kfar Zeita. The government accused rebels of using the chemical.
Last week, opposition activists accused Assad's forces of a new poison gas attack in the Syrian capital and posted footage of four men being treated by medics.
They said this chemical attack, the fourth the opposition has reported this month, was in the Harasta neighborhood of Damascus.