A species of Encephalitozoon has been isolated from the urine of a patient with the acquired immunodeficiency syndrome and maintained in vitro in Madin Darby Canine Kidney cells. When examined by random amplified polymorphic DNA polymerase chain reaction the new isolate was found to differ from E. hellem and to have amplified products in common with murine and canine E. cuniculi. However, it more closely resembled the canine than the murine isolate. Sodium dodecylsulphate polyacrylamide gel electrophoresis differentiated between all three isolates of E. cuniculi, with a band at 42-45 kDa present in the murine isolate only, bands at 52 kDa present in the canine and human isolates but not the murine, and a single band at 60 kDa (murine) and 65 kDa (canine) replaced by two bands at 55 and 70 kDa in the human isolate. The 55 kDa and 70 kDa antigens were also revealed as characteristic bands of the human isolate by Western blotting. The study has thus revealed that the species Encephalitozoon cuniculi is not a homogeneous entity.