Mycobacterium paratuberculosis has been suggested as a causative organism of Crohn's disease. Despite a long-term debate to prove this possibility, the role of this bacteria in the pathogenesis of Crohn's disease is still a subject of controversy. In the present study, serum antibodies (IgG, IgA, and IgM) to the protoplasmic antigen of M. paratuberculosis were quantified in patients with Crohn's disease and in control subjects by using an enzyme-linked immunosorbent assay whose specificity was increased by preabsorbing the sera with cell extracts of Mycobacterium phlei. As compared to normal controls (1/20; 0.062+/-0.022), a significant difference was seen in the antibody-positive prevalence rate and mean values of the serum IgG titer in patients with Crohn's disease (5/13; 0.102+/-0.039) (P < 0.05), but not in patients with ulcerative colitis (2/20; 0.065+/-0.035) and tuberculosis (0/4; 0.053+/-0.008). No significant differences were seen in the antibody-positive prevalence rate and mean values of the serum IgA and IgM titers among the four study groups. These results indicate the unique immune response to M. paratuberculosis in patients with Crohn's disease, suggesting that this organism may play some role in the pathogenesis of Crohn's disease.