A role for mycobacteria in Crohn's disease has been suggested. Isolation of M. paratuberculosis from Crohn's lesions added further support to this relationship. Several studies have investigated the presence of antibodies against M. paratuberculosis in Crohn's patients. While most of these have observed no real differences in the level of antibodies between Crohn's patients and controls, others have reported significant differences. This work was carried out to test that relationship using samples available to us. A total of 197 human sera from two hospitals in the Basque Country, including 66 Crohn's cases, 52 ulcerative colitis cases, 31 active pulmonary tuberculosis cases and 48 healthy controls from blood donors were tested. The sera were absorbed with a suspension of M. phlei (1/1), incubated overnight at room temperature and further diluted up to 1/100 with PBS-TG. The sera were subjected to a conventional ELISA against PPA-3 antigen in duplicate wells, using as conjugate rabbit anti-human IgG labeled with horseradish peroxidase. Statistical analysis showed significant differences in mean IgG reactivity between Crohn's and tuberculosis patients and controls and ulcerative colitis patients. Comparison of the frequency of positive results in Crohn's patients (13.6%) and in controls (0.0%) showed significant differences (p < 0.01), although the highest proportion of positives was found among the tuberculosis group (32.3%). In comparing our results with previous published studies, we submit that the failure to find differences in some of those studies could potentially be explained by the way the results were interpreted. Cross-reactivity among mycobacterial antigens, differences in antigen preparation and testing methodology, changing patterns of sensitization between different populations, and individual variability in the type of disease are known factors influencing the association between humoral immune response and mycobacterial infections that could also account for some contradictory results in studies on serologic responses against mycobacterial antigens in Crohn's disease. Analysis of our results together with all the other published data allowing a positive/negative classification shows a definitely increased reactivity of Crohn's patients against mycobacterial antigens of the M. avium-paratuberculosis group (p < 0.001). This conclusion lends further support to the hypothesis of a role of mycobacteria in the etiology of this disease.