Encephalitozoon intestinalis (Septata intestinalis) is the second most prevalent microsporidian species infecting humans, but it has not been described in other animal species. This investigation examined 10 domestic animal stool samples (8 mammalian, 2 avian) containing spores detected by anti-Encephalitozoon monoclonal antibody immunofluorescence (FA). The presence of E. intestinalis but not Encephalitozoon hellem or Encephalitozoon cuniculi was confirmed in 6 of 8 mammalian stool samples by species-specific FA and polymerase chain reaction. Clusters of spores inside epithelial cells were observed in feces of five mammals (donkey, dog, pig, cow, and goat) using "quick-hot" Gram-chromotrope stain. None of the 10 samples reacted with anti-E. hellem or anti-E. cuniculi sera, nor were they amplified with species-specific primers for E. hellem and E. cuniculi. To our knowledge, this is the first identification of E. intestinalis in animals other than humans. The data shown herein suggest the possibility that E. intestinalis infection may be zoonotic in origin.