Russia, Syrian President Bashar al-Assad's main international backer, denounced the warning and dismissed White House assertions that a strike was being prepared as"unacceptable," raising the tension between Washington and Moscow over the Syrian civil war.
Pentagon spokesman Captain Jeff Davis said the United States had recently seen activity at Shayrat airfield, the same base targeted by a US cruise missile strike on April 6.
Davis said the activity was from "the past day or two." He did not say how the United States collected its intelligence.
"This involved specific aircraft in a specific hangar, both of which we know to be associated with chemical weapons use," Davis said.
The White House said on Monday it appeared the Syrian government was preparing for another chemical weapons attack and it warned Assad that he and his military would "pay a heavy price" if it went ahead.
The US strike on the Shayrat airfield followed the deaths of 87 people in what Washington said was a poison gas attack in rebel-held territory two days earlier. Syria denied it carried out the attack.
The White House said the recent preparations in Syria were similar to actions before the April attack.
But Russia challenged the US intelligence.
"I am not aware of any information about a threat that chemical weapons can be used," Kremlin spokesman Dmitry Peskov told a conference call with reporters on Tuesday.
"Certainly, we consider such threats to the legitimate leadership of the Syrian Arab Republic unacceptable."
Russian officials have privately described the war in Syria as the biggest source of tension between Moscow and Washington, and the April cruise missile strike ordered by US President Donald Trump raised the risk of confrontation between them.
Assad visited a Russian air base at Hmeymim in western Syria on Tuesday, his first visit to the base from which Russian jets have supported his war effort.
Photos circulated showed the Syrian leader in the cockpit of a Russian Sukhoi SU-35 warplane, and inspecting weapons, personnel and armored vehicles at the base near Latakia.
US Warning
The Syrian military and foreign ministry did not immediately comment on the White House warning, though a Syrian state-run television station, al-Ikhbariya, said the White House's allegations were fabricated.
White House officials did not respond to requests for comment on the intelligence that prompted the statement, or on possible US plans if Syria carried out such an attack.
US ambassador to the United Nations Nikki Haley said the Trump administration intended its warning to be aimed not just at Syria's government but also at Russia and Iran, another supporter of Assad.
"I believe that the goal is, at this point, not just to send Assad a message but to send Russia and Iran a message that if this happens again we are putting you on notice," Haley said in a hearing in the US House representatives.
She stressed that the primary US goal in Syria is to fight Islamic State, not to remove Assad.
"I don't see a healthy Syria with Assad in place, but the US priority has and continues to be to fight ISIS," she said.
British Defence Minister Michael Fallon said London would support US action to prevent a chemical weapons attack but that it had not seen the intelligence on which Washington based Monday's statement.
French President Emmanuel Macron agreed with Trump in a phone call on Tuesday to work together to find a common response in case of a new chemical attack in Syria, the French presidency said.
US and allied intelligence officers had for some time identified several sites where they suspected Assad's government may have been hiding newly made chemical weapons from inspectors, said a US official familiar with the intelligence.
That assessment was based in part on the locations, security surrounding the suspect sites and other information which the official declined to describe.
Although the intelligence was not considered conclusive, Washington decided to issue the public warning to the Syrian leadership to try to deter such a strike, said the official, who declined to discuss the issue further.
The number of people killed in suspected chemical attacks is a small portion of the total dead in Syria's civil war - a figure which the Syrian Observatory for Human Rights, a Britain-based war monitor, estimates is close to half a million.
But television footage of victims of April's attack, including children, writhing in agony, caused revulsion across the world.
After the April attack, Trump accused Assad's government of going "beyond a red line" and approved what US officials called a "one-off" strike to deter future chemical attacks.
