Microsporidia are obligate intracellular protozoal pathogens associated with chronic diarrhea in individuals infected with HIV. Direct detection methods for microsporidial spores in stool include chromotrope-based, fluorochrome, and immunofluorescent stains. The authors compared the ability to detect microsporidial spores in 168 stool specimens using two stains: a chromotrope-based modified trichrome stain and a fluorochrome stain, calcofluor white (Cellufluor, Polysciences, Warrington, PA). In addition to being faster and easier to perform, the calcofluor white stain was found to be more sensitive than the chromotrope-based stain, as 6 of 24 specimens positive by calcofluor white were negative by the chromotrope-based stain on initial smear evaluation. Repeat examination confirmed these six as being positive. To evaluate the specificity of the calcofluor white stain, 20 formalin-fixed stool specimens (5 positive and 15 negative for microsporidial spores) were evaluated in blinded fashion by two affiliated clinical laboratories using their own formulations of calcofluor white. A single discrepant result (falsely positive) was reported from one laboratory. The use of the calcofluor white stain is recommended as a simple and highly sensitive screening procedure for the detection of microsporidial spores in stool specimens.