Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) with tissue granuloma and histopathological alteration that resembles aspects in tuberculosis, leprosy, and paratuberculosis. Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of paratuberculosis, with a suspected role in the etiology of CD. We investigated the presence of MAP DNA in 31 surgical tissue samples from 20 subjects using fluorescence in situ hybridization (FISH) with the aid of confocal scanning laser microscopy and nested polymerase chain reaction (PCR) using the IS900 sequence unique to MAP. MAP DNA was detected by PCR in tissue from 10 of 12 (83%) patients with CD: 7/12 (58%) in inflamed, 6/11 (55%) in noninflamed and in 10 (83%) of either tissue and by FISH in 8 of 12 (67%) patients with CD: 7 of 12 (58%) in inflamed, 4 of 11 (36%) in noninflamed, and in 8(67%) of either tissue. In non-IBD subjects, MAP DNA was detected in the tissue of only 1 of 6 patients (17%) by PCR and 0 of 6 patients (0%) by FISH. MAP DNA was identified by PCR in inflamed tissue from 2 of 2 patients with ulcerative colitis. The detection of MAP DNA by either technique in tissue from subjects with CD is significant compared with non-IBD subjects (P < 0.005). Identification of MAP DNA in both inflamed and noninflamed tissue by both techniques suggests that MAP infection in patients with CD may be systemic. The data add more evidence toward a possible association of MAP in the pathogenesis of CD.