A previously unrecognized Mycobacterium species was isolated from two patients with Crohn's disease. The organism is an acid-fast, mycobactin-dependent Mycobacterium that has characteristics which do not conform to any of the presently recognized species. It belongs to the Runyon group III mycobacteria and is most closely related to Mycobacterium paratuberculosis. Animal inoculation revealed pathogenicity for mice when injected intravenously or intraperitoneally, but not for rats, guinea pigs, rabbits, or chickens. The mice developed hepatic and splenic granulomas which contained numerous acid-fast mycobacteria. A 7-day-old goat which was inoculated orally with 50 mg of the organism developed both humoral and cell-mediated immunologic responses in two to three weeks and granulomatous disease of the distal small intestine, with noncaseating tuberculoid granulomas in five months. Acid-fast bacilli were not demonstrable in sections of the intestine, but a single organism was seen in each of two microgranulomas of the mesenteric lymph node. The Mycobacterium species was reisolated from the lymph node but not from intestine. Our findings raise the possibility that a Mycobacterium plays an etiologic role in at least some cases of Crohn's disease.