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"Turkey must choose," US vice president Mike Pence said. "Does it want to remain a critical partner in the most successful military alliance in history, or does it want to risk the security of that partnership by making such reckless decisions that undermine our alliance?"
His remarks came after Turkey insisted on Wednesday the Russian deal was already done.
Washington fears the Russian S-400 missile system would compromise the security of its F-35 fighter jets.
Speaking at a Nato 70th anniversary event, Mr Pence added: "We've also made it clear that we will not stand idly by while Nato allies purchase weapons from our adversaries that threaten the cohesion of our alliance."
Turkey retaliated promptly, with its vice president Fuat Oktay tweeting: "The United States must choose. Does it want to remain Turkey's ally or risk our friendship by joining forces with terrorists to undermine its Nato ally's defence against its enemies?"
The US' support to the Kurdish-led Syrian Democratic Forces (SDF) fighting against Islamic State in Syria has infuriated Turkey, which views the SDF as a terrorist organisation.
Washington has however warned proceeding with the deal could result in US sanctions and the expulsion of Turkey from the F-35 fighter jet programme.
On Monday, the Pentagon stopped the delivery of F-35 parts and manuals to Turkey.
The US has offered to sell Turkey the American-made Patriot missile defence system, but Turkish foreign minister Mevlut Cavusoglu said it could not be delivered on time.
"We couldn't get it for 10 years," Mr Cavusoglu said at the Atlantic Council on Wednesday morning. "That's why we had to buy from Russia. And we tried to buy from other allies as well. It didn't work. So it is an urgent need of Turkey. I mean, we need air defence systems urgently in Turkey."
Ankara says it needs the missile system to defend itself, as Turkey faces threats from Kurdish and Islamist militants at home and also from conflicts in neighbouring Syria and Iraq.
The US and other Nato allies have repeatedly complained about the purchase of the S-400 system, saying it is not compatible with other allied systems and would represent a threat to the F-35.
Officials have said Turkey's acquisition of both US and Russian systems could give Moscow access to sophisticated American technology and allow it to find ways to counter the stealth fighter jet.
Additional reporting by agencies