The detection of microsporidial DNA by the polymerase chain reaction (PCR) has been suggested as an alternative or supplement to conventional microscopic methods. However, the relative merits of these techniques remain uncertain. In the present study, clinical specimens of different origin (stool, urine, sputum, nasal discharge, and cerebrospinal fluid) containing four different microsporidial species were blinded after microscopic examination and analyzed by PCR and subsequent restriction fragment length polymorphism (RFLP) to determine the respective species. Thirty-four specimens from 31 patients were evaluated, 16 of which were positive and 18 negative by microscopic examination; PCR detection of microsporidia produced identical results in 82% (28/34) of these specimens. Four samples were microscopically negative, PCR-positive, and two were microscopically positive, PCR-negative. Species determination by RFLP analysis of the amplified product was accurate for all isolates containing Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi compared with microscopic identification of the genus Enterocytozoon or molecular analysis of Encephalitozoon species after in vitro culture. Therefore, PCR-RFLP is useful for the rapid detection and differentiation of microsporidian spores in clinical specimens.