Microsporidia spores, identified as Encephalitozoon cuniculi (CDC: V282), were isolated from the urine of a patient with acquired immunodeficiency syndrome and disseminated microsporidiosis, established in continuous culture on monkey kidney cells (E6), and antiserum was produced in rabbits. Immunoblot studies that used the patient serum and the rabbit sera against CDC:V282, Encephalitozoon hellem (CDC:0291:V213), and Encephalitozoon intestinalis (CDC:V297) revealed that CDC:V282 and the rabbit isolate of E. cuniculi (ECLD) reacted intensely with the patient's serum and the rabbit anti-CDC:V282, producing a number of bands ranging from 200 to 15 kDa. By contrast, the heterologous antigens (CDC:0291:V213 and CDCV297) reacted minimally. Both CDC:V282 and ECLD isolates of E. cuniculi reacted minimally with the rabbit anti-E. hellem and the rabbit anti-E. intestinalis sera. In the immunofluorescence test, performed on the lung biopsy section of the patient, the rabbit anti-CDC:V282 serum reacted extensively with the spores in the tissue section and produced bright apple green fluorescence. These studies demonstrated that the human (CDC:282) and the rabbit (ECLD) isolates of E. cuniculi were similar in their antigenic profiles but differed considerably from E. hellem and E. intestinalis, and that the patient's serum reacted specifically, strongly, and with equal intensity, with the 2 isolates of E. cuniculi.