Spokeswoman Heather Nauert said the two Cubans were asked to leave the US on May 23 after Americans in Cuba "reported incidents which have caused a variety of physical symptoms," causing them to leave the island.
Nauert said the first of the incidents was reported in late 2016 and that they had continued. She would not say what the symptoms were except that they were not life-threatening.
However, other US officials said that the symptoms included hearing loss.
One person familiar with the US investigation said investigators were looking into whether elements of the Cuban government placed sonic devices that produce non-audible sound inside or outside the residences of roughly five US Embassy staffers with the intent of deafening them. That individual and the US officials weren't authorized to discuss the investigation publicly and spoke on condition of anonymity.
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Cuba employs a massive state security apparatus that keeps hundreds and possibly thousands of people under constant surveillance.
However, the person familiar with the US probe said investigators were looking into whether the individuals were harmed outside the regular chain of command of the Cuban government.
The officials said the staffers all arrived in Havana in the summer of 2016. Like all foreign diplomats in Cuba, they lived in housing owned and maintained by the Cuban government.
In the fall, officials said the affected diplomats and their spouses began to experience symptoms of hearing loss so severe and puzzling that an investigation was launched, and it was determined that they were at risk. They were allowed to leave Cuba, the officials said.
Although she would not provide details, Nauert said that investigators did not yet have a definitive explanation for the incidents but stressed they take them "very seriously" and are working to determine their "cause and impact."
She said the department had reminded Cuba of its international obligation to protect foreign diplomats. Harassment of American diplomats in Cuba is not uncommon and dates to the restoration of limited ties with the communist government in the 1970s.