The microsporidium Enterocytozoon bieneusi is closely linked to wasting and diarrhea in a high proportion of individuals with AIDS. However, its relative contribution to disease is uncertain because diagnosis until recently depended on procedures involving endoscopy. A sensitive PCR technique which amplifies a fragment of the small-subunit rRNA gene of E. bieneusi from formalin-fixed stool samples was developed. Of 80 formalin-fixed stool samples collected from 74 Zimbabweans and 6 U.S. patients who were human immunodeficiency virus positive, 50% tested positive for E. bieneusi by PCR, whereas 24% tested positive for E. bieneusi by light microscopy of trichrome-stained fecal smears. In addition, we describe an in situ hybridization technique which detected and identified E. bieneusi as the causative agent in all six intestinal biopsy specimens tested. Both the PCR and in situ hybridization procedures are sensitive diagnostic tools which will complement currently available techniques and enable the differentiation of E. bieneusi from other microsporidia to be made.