There are about 100 US nationals "operating inside of Syria" but it was unclear which rebel groups they were associated with, spokesman Colonel Steven Warren said.
"We believe there are maybe a dozen that are with ISIL (IS militants)," he said.
Senior US officials have voiced concern at the presence of foreign fighters among the Sunni extremists who hold Western passports, potentially enabling them to return from the battlefield prepared to carry out terror attacks in Europe or the United States.
At least one American fighting for IS was killed in combat in Syria, officials say, while authorities were investigating if a second US national also died as an IS recruit.
The Federal Bureau of Investigation has arrested several people trying to travel from the United States to Syria to support the IS militants, Matthew Olsen, director of the US National Counter-Terrorism Center, said Wednesday.
While there was no indication the IS extremists were plotting an imminent attack on the United States, there was a danger that a sympathizer of the group could carry out a "limited" attack on American soil, according to Olsen.
The United Nations and human rights groups cite growing evidence of a litany of atrocities committed by IS fighters in northern Iraq and eastern Syria against ethnic and religious minorities.
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