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EU Trade Commissioner Maros Sefcovic warned that Europeans "stand ready to defend our interests"
The European Union's trade chief said the 27-member bloc is committed to securing a trade deal with the US based on "respect" not "threats".
It comes after US President Donald Trump threatened to slap a 50% tariff on all goods sent to the United States from the EU.
"The EU's fully engaged, committed to securing a deal that works for both," EU Trade Commissioner Maros Sefcovic said after a call with US Trade Representative Jamieson Greer and Commerce Secretary Howard Lutnick.
"EU-US trade is unmatched & must be guided by mutual respect, not threats. We stand ready to defend our interests."
Earlier on Friday, Trump expressed impatience with the pace of ongoing EU-US trade negotiations, saying his plan to raise tariffs on 1 June was set.
Posting on his Truth Social platform, Trump said: "Our discussions with them (the EU) are going nowhere," adding that there would be no tariffs for products built or manufactured in the US.
"I'm not looking for a deal - we've set the deal," he told reporters later, before immediately adding that a big investment in the US by a European company might make him open to a delay.
The EU is one of the Washington's largest trading partners, sending more than $600bn (€528bn; £443bn) in goods last year and buying $370bn worth, US government figures show.
Reacting to Trump's threats, European governments have warned that higher tariffs would be damaging to both sides.
"We do not need to go down this road. Negotiations are the best and only sustainable way forward," Ireland's Taoiseach (Prime Minister) Micheál Martin said.
"We are maintaining the same line: de-escalation, but we are ready to respond," said French Foreign Minister Laurent Saint-Martin.
German Economy Minister Katherina Reiche said that "we must do everything to ensure that the European Commission reaches a negotiated solution with the United States".
Dutch Prime Minister Dick Schoof told reporters that he backed the EU's strategy in trade talks and "we have seen before that tariffs can go up and down in talks with the US".
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US President Donald Trump in April announced what he called "reciprocal tariffs" on goods from countries around the world
Last month, Trump announced a 20% tariff on most EU goods but had halved it to 10% until 8 July to allow time for more negotiations.
Trump's complaints about Europe have focused on its uneven trade relationship, as the EU sells more goods to the US than it buys from America.
He blames this trade deficit on policies that he says are unfair to American companies, and he has specifically raised concerns about policies related to cars and agricultural products.
Trump also warned Apple that he would impose a 25% import tax "at least" on iPhones not manufactured in America, later widening the threat to any smartphone.
Shares in the US and EU fell on Friday after the latest threats, with the S&P 500 down about 0.7% and Germany's Dax and France's Cac 40 ending the day down more than 1.5%.