Crohn's disease is an inflammatory bowel disease of unknown etiology that shares clinical and histological features with mycobacterial infections. Recent data using improved culture techniques and specific PCR-based assays have strengthened the association of M. paratuberculosis with Crohn's disease. To provide more direct evidence of an etiological association, we studied the relationship between M. paratuberculosis and the immune system at the humoral immune response level. We used sera from patients with Crohn's disease, tuberculosis, leprosy, ulcerative colitis, sarcoidosis, and non-inflammatory bowel disease controls (patients with gastric and duodenal ulcer) in SDS-immunoblots of M. para sonicate and recombinant clones selected from an M. paratuberculosis expression library to study the humoral immune response to M. para antigens. Both Crohn's disease and control sera reacted with M. paratuberculosis sonicate without an identifiable specific pattern. However, sera from 8 of 10 Crohn's disease, 3 of 4 tuberculosis, all 6 leprosy and 5 sarcoidosis patients reacted with a recombinant clone expresses a 38K protein (p38). Only 4 of 10 ulcerative colitis and 4 of the 15 controls reacted with p38. The difference between reactivity between Crohn's disease and ulcerative colitis, controls or both was significant (p < 0.05). The fact that sera from Crohn's disease patients reacted with the same high frequency as the sera with known mycobacterial infection is further evidence to support the hypothesis of the mycobacterial etiology in Crohn's disease and perhaps sarcoidosis.