In the past several years, microsporidia have become recognized as another important group of opportunistic infections of immunocompromised patients, especially those with AIDS. Enteric infections with the noncultivatable microsporidian parasite Enterocytozoon bieneusi have been diagnosed from AIDS patients with chronic diarrhea, malabsorption, and wasting. The incidence of infection and mechanism of transmission of these organisms in humans is unknown. Several recent tests for human pathogens have been developed using rRNA genes as diagnostic probes. Using the polymerase chain reaction and conserved regions of the small subunit rRNA (SSU-rRNA) gene, the SSU-rRNA gene of E. bieneusi was successfully cloned and subsequently sequenced. Amplification of E. bieneusi rRNA could be demonstrated from intestinal biopsies from HIV-1-infected patients infected with E. bieneusi but not from intestinal biopsies from noninfected patients. This cloned SSU-rRNA gene was used to develop improved probes for detection of E. bieneusi in tissue of infected patients.