During the months of June and July 1998, stool samples from 341 hospitalized patients (mean age, 32.7 +/- 16.3 years; range, 1-86 years) from Kathmandu, Nepal, were screened for the prevalence of Encephalitozoon sp. by use of anti-Encephalitozoon sp. monoclonal antibody 3B6-based immunofluorescence assay. The cross-sectional study revealed the presence of Encephalitozoon spores in 0.6% (2 of 341) patients. By use of direct microscopic examination, 27% (93 of 341) of patients were diagnosed with various gastrointestinal pathogens, among which Ascaris lumbricoides and Ancylostoma duodenale were the most commonly found, with prevalence rates of 8.8% (30 of 341) and 7.6% (26 of 341), respectively. To our knowledge, this is the first study to report the presence of Encephalitozoon sp. among humans in Nepal.