Microsporidia are protozoa parasites responsible for significant gastrointestinal disease in patients infected with human immunodeficiency virus. We evaluated a PCR assay of stool samples, duodenal aspirates, and biopsy specimens from patients with Enterocytozoon bieneusi infection. A 210-bp DNA fragment of the unique rRNA intergenic spacer could be amplified from all samples infected with E. bieneusi, but no amplification was seen by using DNA purified from samples with Septata intestinalis or other parasites and from negative control human cells. These results suggest that the PCR in stool samples may be a useful tool for the diagnosis of intestinal microsporidiosis in patients with AIDS.