"We intend to investigate thoroughly exactly what happened," Obama told reporters during a joint news conference in Jerusalem with Israeli Prime Minister Benjamin Netanyahu.
The president said he was "deeply skeptical" of Syrian government claims that the opposition used chemical weapons.
Obama has previously said Syria's use of chemical weapons would cross a "red line."
"We have been very clear to the Assad regime -- but also to other players on the ground -- that a red line for us is, we start seeing a whole bunch of chemical weapons moving around or being utilized," he told reporters. "That would change my calculus; that would change my equation."
Intelligence agencies pore over the evidence
Intelligence officials around the world were investigating the accusations, U.S. officials told CNN on Wednesday.
Investigators were talking to rebels and defectors, poring over medical intelligence regarding symptoms reported by doctors and looking at satellite imagery used to track missiles launched and chemical weapons movements, the officials said on condition of anonymity. They were not authorized to release details to the media.
A spokesman for Netanyahu, Mark Regev, told CNN that Israeli officials had no confirmation that chemical weapons had been used.
But Regev's comments did not square with those of Israeli Justice Minister Tzipi Livni.
"It is clear for us here in Israel" that chemical weapons have been used in Syria, and an international response should be on the table, Livni told CNN in an exclusive interview from her home in Tel Aviv.
Livni wouldn't say whether there is evidence the Syrian government has directed the use of any chemical weapons.
But she said the development would pose a direct threat to Israel, which shares a border with Syria.
Their concerns centered on an attack Tuesday in Khan al-Asal in the northern province of Aleppo. State-run media blamed rebels for the attack, which it said killed 25 people and injured more than 110 others.
Rebels say they have no chemical weapons
On Wednesday, Syria's U.N. ambassador, Bashar Ja'afari, said the Syrian government has asked Secretary-General Ban Ki-moon to open an investigation into the alleged use of chemical weapons by "terrorist groups," which is how the government refers to rebels.
But the opposition Free Syrian Army said rebels don't have access to chemical weapons and blamed the casualties on a government missile.
Separately, an opposition group said the government attacked the rural Damascus suburb of Ateibeh with "chemical rockets," causing an unspecified number of deaths along with cases of suffocation, nausea and hysteria. There was no immediate government response.
The reports ignited a firestorm of reactions, with Russia slamming the rebels and some U.S. lawmakers saying that Washington might need to take action against the Syrian government.
Observers: Images are not consistent with a chemical weapons attack
But images posted by Syrian state-run media of the aftermath of the Aleppo incident, which the government blamed on rebels, are not consistent with a chemical weapon attack, some observers said.