We describe an in vitro culture technique for a microsporidian isolated from the corneal biopsy of an HIV-infected patient. The corneal biopsy was inoculated into a monolayer culture of fibroblasts derived from newborn mouse brain and incubated at 37 degrees C in an atmosphere of 5% CO2. Minimum essential medium supplemented with 2% fetal bovine serum appeared to be an optimum medium for growth and maintenance of the parasite and for production of large numbers of spores. This microsporidian was identified as Trachipleistophora anthropophthera based on ultrastructural features. It forms two types of sporophorous vesicles and two types of spores simultaneously: polysporous vesicle type I with eight or more oval spores, 3.7-4.0 microm by 2.0-2.3 microm, and bisporous vesicle type II with two round spores, 1.7-2.2 microm by 1.6-2.0 microm in size.