Bolivia says agents of the U.S. Drug Enforcement Administration (DEA) based locally must leave in three months.
The Bolivian government issued the deadline Tuesday, days after leftist President Evo Morales suspended their work, accusing them of spying and supporting anti-government protests.
President Morales alleges the DEA officials financed "criminal groups" so they could act against Bolivian government officials, including the president.
Mr. Morales accused the DEA of supporting groups that took part in violent political protests in September. U.S. officials rejected the accusations.
The United States recently suspended Bolivia's trade preferences. The deal lowers tariffs on Andean nations that help the United States fight drugs. The U.S. has accused Bolivia of failing to meet its obligations to combat drug trafficking.
In October, U.S. President George Bush signed a six-month extension of the Andean Trade Preference Act, which extends privileges to Colombia, Ecuador and Peru.
Some information for this report was provided by AFP and Reuters.