Nicolas Maduro, the Venezuelan president, has accused Washington of plotting with anti-government protesters and expelled three US diplomats in retaliation.
Maduro's ordered the expulsions on Monday, the same day that opposition leader Leopoldo Lopez called for a mass rally on Tuesday and challenged the government to arrest him at the event.
"I have ordered the foreign ministry to proceed with declaring those three consular officials persona non grata and expelling them from the country. Let them go conspire in Washington!" Maduro said in a nationally broadcast address.
The US State Department denied the charges, calling them "baseless and false" and urging the Venezuelan government to engage the opposition in "meaningful dialogue."
Maduro said that the US diplomats, who have not been named, had met students involved in anti-government protests under the pretence of offering them "visas to the United States".
Venezuela's Foreign Minister Elias Jaua said the three consular staff had used visa visits to universities as cover for promoting opposition protests by students, adding they had 48 hours to leave the country.
The demonstrations, which have energised the opposition but show few signs of ousting President Maduro, continued on Monday with scattered protests in the capital, Caracas, and various provincial cities.
Nearly two weeks of anti-government protests lead by students have become the biggest challenge to Venezuela's socialist rulers since the death of longtime leader Hugo Chavez last year.
Venezuela is mired in a deep economic crisis that critics blame on policies that Maduro largely inherited from Chavez.
Strict controls on currency and prices have fuelled inflation and emptied store shelves.
In late September Maduro kicked out three other US diplomats, including the charge d'affairs, Kelly Keiderling, on accusations of conspiring with government opponents.
The US on Monday rejected as "baseless and false" accusations by Venezuela.
"We support human rights and fundamental freedoms - including freedom of expression and of peaceful assembly - in Venezuela as we do in countries around the world," State Department spokeswoman Jen Psaki said.
"But as we have long said, Venezuela's political future is for the Venezuelan people to decide."
The two countries have had no ambassadors since 2010.
A Venezuelan foreign ministry statement also said that Maduro's government "flatly rejects" remarks on Saturday by the US secretary of state, John Kerry on the use of Venezuelan security forces to suppress protests.
Kerry's statement was "yet another manoeuvre" by Washington to "legitimise attempts to destabilise the Venezuelan democracy”, the ministry said.
Henrique Capriles, an opposition leader who narrowly lost to Maduro in last year's election, has given warning that now is not the time to try to force the government out of power.
Three people were killed on Wednesday when an opposition demonstration turned violent, setting off days of clashes in the streets between protesters and security forces.
The government of Maduro has accused right-wing infiltrators of fomenting the violence. But leaders of the student movement blamed groups of hooded assailants backed by the government.