A 46-year-old human immunodeficiency virus-infected Swiss citizen living in Tanzania presented with respiratory, abdominal, and urogenital complaints. Microsporidial spores were isolated from urine and a sinunasal aspirate and were propagated in MRC-5 cell cultures. Western blot analysis and riboprinting identified the sinunasal isolate as Encephalitozoon hellem. Electron microscopic investigation of the urine isolate revealed spores with diplokaryotic nuclei and five to six isofilar coils of the polar tube and sporonts with two or three diplokarya. All stages were enveloped by two membranes, corresponding to a cisterna of host endoplasmic reticulum studded with ribosomes. These characteristics have been described for the genus Vittaforma. Western blot analysis of this isolate revealed a banding pattern identical to that of the Vittaforma corneae reference isolate. Part of the small subunit rRNA gene was amplified, sequenced (239 base pairs), and found to be identical to that of V. corneae. This is the second isolation of V. corneae and the first description of urinary tract infection due to V. corneae in a patient with AIDS.