Enterocytozoon bieneusi is the most frequently reported microsporidial infection of humans. In patients with the acquired immunodeficiency syndrome, Enterocytozoon infects the lining epithelial cells of the small intestine, hepatobiliary tract, and gallbladder. Because Enterocytozoon has been thought to be limited to infecting lining epithelial cells, the mechanism of spread of E bieneusi within the intestine, to the biliary tract, and, in two case reports, to distant organs remains unknown. This report describes a patient with acquired immunodeficiency syndrome and intestinal microsporidiosis due to E bieneusi. Histopathologic examination of well-oriented biopsies from the duodenum and jejunum revealed both intra- and extracellular spores of Enterocytozoon extending deeply into the lamina propria, where they were located adjacent to capillaries. The patient has not developed disseminated disease 20 months after the initial diagnosis. In this patient, the demonstration of E bieneusi spores in extraepithelial tissues does not appear to be associated with development of subsequent systemic infection.