A new species of microsporidian, Septata intestinalis, was recently recognized as an opportunistic pathogen of AIDS patients. In this study, it was cultured from the nasopharyngeal aspirate of a human immunodeficiency virus type 1-infected patient with disseminated microsporidiosis. In human embryonic lung cells exposed to S. intestinalis, a cytopathic effect appeared within 28 days as foci of rounded up cells. Thin-section electron microscopy showed a variety of developmental stages of the microsporidium within parasitophorous vacuoles. In monocyte-derived macrophages, evidence of infection and development of the parasite was demonstrated by light and electron microscopy. In both infected human embryonic lung cells and monocyte-derived macrophages, a network of sepia separated individual spores. Partial sequencing of the RNA small-subunit gene (16S rDNA gene) confirmed the identity of this parasite as S. intestinalis. This is the first report of the isolation of S. intestinalis in vitro and provides evidence that the parasite can be disseminated by macrophages.