The etiology of Crohn's disease remains unknown with inflammatory, infectious, and/or genetic causes suspected. Granulomatous inflammation is a characteristic feature of the disorder, resembling the tissue response to mycobacterium. Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent in Johne's disease, a chronic ulcerative intestinal condition in cattle, and has been implicated as a likely candidate. We carefully microdissected the granulomas from the paraffin-embedded resection specimens of 18 patients with well-established Crohn's disease. The DNA obtained was PCR amplified for the IS900 and IS1311 repeat elements of MAP, PCR product size maintained at 101 and 124 base pairs, respectively. Archival tissue from bovine Johne's disease was used as a positive control. MAP-specific DNA, confirmed by sequencing and comparison with prototype strain sequence, was appropriately amplified from the positive control. None of the Crohn's disease cases yielded a positive amplification product, failing to support a role for the organism in the pathogenesis of this illness.