Microsporidia are the suspected etiology of diarrhea in up to 30% of immunocompromised patients with AIDS. Epidemiological investigation of these organisms has been hampered by the lack of easy methods of detection. Currently, definitive identification requires small bowel biopsy and transmission electron microscopy (TEM) to confirm the species of microsporidia involved. We describe a method for the detection of microsporidia in stool specimens that utilizes polymerase chain reaction (PCR) analysis with use of primers V1 and EB450 based on the small-subunit rRNA gene of Enterocytozoon bieneusi. A guanidium thiocyanate (GuSCN) method was utilized to extract microsporidian DNA from feces. Consistent amplification was detected in stool samples from three patients with TEM-confirmed microsporidiosis due to E. bieneusi by means of these techniques. Optimization of this PCR reaction revealed that the sensitivity of the reaction was increased by the addition of dimethyl sulfoxide. This PCR method allows for detection of E. bieneusi in stool specimens and will be applied to epidemiological investigations of this organism in the future.