Detection of microsporidia in clinical specimens has relied on electron microscopy, histology, or staining. This article describes further alterations to the modified trichrome staining method which make it easier to identify microsporidial spores. The changes are a decrease in the phosphotungstic acid level and the substitution of a colorfast counterstain, aniline blue, for the fast green of the original stain. The modified stain provides good contrast between microsporidial spores and background material including human and fungal cells. Stool specimens from 139 human immunodeficiency virus-seropositive patients revealed that 5 patients were infected with Enterocytozoon bieneusi and 6 patients had larger spores. Thin-section electron microscopy of the larger spores showed a structure consistent with that of either Encephalitozoon or Septata species. Three of the patients with Encephalitozoon- or Septata-like species had disseminated infection, with spores detected in nasopharyngeal aspirates and urine samples.