The microsporidian Encephalitozoon hellem is being reported with increasing frequency in HIV-positive subjects, as an agent of disseminated microsporidiosis without involving the gastrointestinal tract. We describe a case of pulmonary microsporidiosis in a 27-year-old Italian man with AIDS who developed fever, cough, and dyspnea. A chest X-ray showed multiple bilateral pulmonary opacities and mediastinal lymph-node enlargement. Stained smears of bronchoalveolar lavage sediment showed oval structures consistent with microsporidian spores. Viral, bacterial and fungal cultures were repeatedly negative, whereas microsporidia were successfully cultured in human and bovine fibroblast cell lines. Analysis of electron micrographs indicated that the isolate belonged to the genus Encephalitozoon. Based on further immunological, biochemical and molecular studies it was characterized as E. hellem. Even though a temporary improvement with albendazole therapy was noticed, the patient deteriorated clinically and died of severe respiratory distress.