Recent data using improved cultural, Molecular, and serological techniques have strengthened the association of Mycobacterium paratuberculosis with Crohn's disease, an inflammatory bowel disease (IBD) with unknown etiology. To provide more evidence of an etiological association, antibody reactivities of Crohn's disease patients were tested by immunoblotting against M. paratuberculosis-recombinant antigens. A clone containing a 1,402-bp insert and expressing a 36K-antigen (p36) was analyzed. No homology was found between the deduced amino acid sequence of p36 and any protein sequences compiled in the GenBank indicating that p36 is a novel mycobacterial protein. The reactivity of 199 serum samples was tested against the p36 by immunoblotting technique. Sera from 77 of 89 (86.5%) Crohn's disease patients and 16 of 18 (89%) sera from patients with tuberculosis and leprosy reacted with p36 compared to 5 of 42 (12%) ulcerative colitis and non-IBD control sera (p < 0.0001). In addition, p36 reacted to all sera from 10 normal controls that were Bacillus Calmette-Guerin (BCG)-immunized and only to 10% of 40 normal controls that were not BCG-immunized. The fact that sera from Crohn's disease patients reacted to p36 with the same high frequency as the sera from patients that were exposed to mycobacterial antigens further supports the hypothesis of the mycobacterial etiology in Crohn's disease.