To assess the importance of microsporidiosis of the small intestine in the pathogenesis of chronic diarrhoea in HIV-1-infected individuals, duodenal biopsy samples from the following three patient groups were prospectively evaluated for bacterial, viral, and parasitic pathogens by standard methods, and for microsporidia by light microscopy: 55 consecutive HIV-1-antibody-positive subjects with unexplained diarrhoea of at least 3 weeks duration (group A); 38 HIV-1-seropositive subjects without diarrhoea (group B) who consecutively underwent upper gastrointestinal endoscopy for various reasons; and 7 patients without known risk factors for HIV infection with chronic unexplained diarrhoea (group C). In groups A and B most subjects had had previous AIDS-defining opportunistic infections and the median peripheral blood CD4 lymphocyte count was less than 0.1 x 10(9)/l. Microsporidia were detected as the single pathogen in 15 of the group A compared with 1 (in whom diarrhoea subsequently developed) of the group B patients (p = 0.001) and none of the group C patients. With the exception of 4 of the group A patients, no other intestinal pathogens were identified in any of the patients. The median peripheral blood CD4 count was significantly lower in patients with detectable microsporidia than in those without microsporidiosis (0.03 x 10(9)/l vs 0.06 x 10(9)/l; p = 0.03); in all patients with microsporidiosis, the CD4 count was equal to or less than 0.1 x 10(9)/l. 13 patients with microsporidiosis were treated with metronidazole, in 10 of whom treatment led to a substantial improvement or disappearance of diarrhoea within days of starting therapy, but did not result in eradication of the parasite in the 5 patients who underwent repeat biopsy. The findings suggest that small-intestinal microsporidiosis is an important cause of chronic unexplained diarrhoea in HIV-1-infected individuals with pronounced cellular immune deficiency. This infection should therefore be added to the list of AIDS-defining opportunistic infections.