A new human microsporidian was isolated from cultures of Madin-Darby canine kidney cells incubated with conjunctival scrapings or corneal tissues from three AIDS patients with keratoconjunctivitis. The three isolates were morphologically similar to Encephalitozoon cuniculi. The spores averaged 1 x 1.5-2.0 microns, had six to eight polar filament coils, displayed monokaryotic nuclei, and possessed relatively thick endospores with irregularly shaped exospores. Organisms developed within a parasitophorous vacuole. By SDS-PAGE analysis, the three isolates appeared to be identical but were different from E. cuniculi. Identical banding patterns on Western blots of the three isolates were expressed by each patient's serum. By Western immunoblotting, murine antisera to E. cuniculi reacted to several antigens of the new AIDS-related microsporidian, whereas murine antisera bound weakly to Nosema corneum. The name Encephalitozoon hellem (n. sp.) is proposed to identify this new human microsporidian.