Microsporidia are emerging as opportunistic pathogens in patients with acquired immunodeficiency syndrome (AIDS). Enterocytozoon bieneusi is the most commonly reported microsporidium that is detected in gastrointestinal specimens. This report describes an in situ hybridization technique with a 30-base specific synthetic DNA probe for detection of E bieneusi by light microscopy. Formalin-fixed paraffin-embedded duodenal biopsy specimens from three patients with AIDS, chronic diarrhea, and E bieneusi infection confirmed by electron microscopy were used in this study. Light microscopic examination after colorimetric detection allowed the identification of different stages of the pathogen's life cycle in the cytoplasm of enterocytes. No cross-reactivity was noted between the probe and human DNA. Our study underscores the applicability of a synthetic-labeled oligonucleotide for the detection and identification of E bieneusi in clinical samples.