In order to assess the impact of highly active antiretroviral therapy (HAART) on the frequency and etiology of fever of unknown origin in HIV-infected patients, a retrospective study was performed on cases of fever of unknown origin observed from January 1997 to December 1999 in seven hospitals in five cities in Andalusia, Spain. During the period specified, a total of 4,858 HIV-infected patients receiving HAART and 2,787 HIV-infected individuals not receiving HAART were studied. The frequency of fever of unknown origin was 0.6% in patients receiving HAART and 3% in non-HAART patients (P=0.001). Human immunodeficiency virus infection was the only cause of fever found more frequently in the non-HAART patient group (P=0.07). The study findings suggest that the use of HAART has reduced the frequency of fever of unknown origin in HIV-infected patients, but the etiology of the condition remains mostly unchanged.