To evaluate the sensitivity of autofluorescence for detection of Isospora oocysts, wet preparations of 192 stool samples from patients with chronic diarrhea were examined by fluorescence microscopy and by light microscopy after iodine staining used for routine screening for ova and parasites. Silicon-chambered glass coverslips were used for fluorescence microscopy. Isospora oocysts were detected in 46 iodine-stained concentrated stool samples; 91 samples were positive by autofluorescence. According to the maximum likelihood estimates, examination by fluorescence (95.7%; 95% confidence interval [CI], 85.2-99.5) was significantly more sensitive than iodine staining (48.4%; 95% CI, 37.7-59.1). Examination for autofluorescence is a simple, highly sensitive, inexpensive, and easily applicable method to detect Isospora spp. oocysts in feces.