Enterocytozoon bieneusi is a newly described microsporidia in humans thought to be responsible for chronic diarrhoea in acquired immunodeficiency syndrome (AIDS) patients. The epidemiology of this parasite is still unknown; it could be a strictly opportunistic agent or a human enteropathogen. E. bieneusi spores were searched for in stool smears of two populations using a modified chromotrope 2R staining. The first population consisted of 60 patients infected by the human immunodeficiency virus (HIV) and the second of 990 children aged from one month to six years consulting two primary care centers in Niamey, Niger. E. bieneusi spores were found in 4 out of the 60 HIV-positive patients (7%). These 4 patients belonged to a subgroup of 35 patients with < 50 CD4 cells/microliters. Out of 990 children, 8 shed E. bieneusi spores in their stools; the presence of spores was not associated with a particular clinical phenotype (diarrhoea, fever, dehydration, vomiting). Although HIV status could not be evaluated, the HIV prevalence rate among women consulting the same care centers was low (0.5%) and it is therefore unlikely that all eight children were HIV-infected. The results show for the first time that E. bieneusi can infest HIV-negative subjects. Microsporidiosis is frequent in AIDS patients with low CD4 cell counts. Further work is needed to define the prevalence and the possible pathogenic effect of E. bieneusi in immunocompetent subjects.