On Sunday, an assembly of Afghan elders, known as the Loya Jirga, endorsed the security pact, but Karzai suggested he might not sign it until after national elections next spring.
The impasse strengthens questions about whether any U.S. and NATO troops will remain after the end of next year in Afghanistan, which faces a still-potent insurgency waged by Taliban militants and is still training its own military.
Karzai's defiance has surprised the many who had attended the Loya Jirga, which he had proclaimed would have the final word on the security deal.
A senior politician in Kabul said it appeared that Karzai's reluctance to let the deal go through stemmed from his eagerness to keep his hands on the levers of power in the run-up to a presidential election in April, when he is due to stand down.
"He is now in confrontation with his own nation as well as the United States," said the politician, who asked not to be named.
He added that the president's demand for no U.S. meddling in the coming election suggested that Karzai could be looking to ensure he has room to influence the outcome himself.