The previously described (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol., 31:947-954, 1993) a362 recombinant polypeptide of Mycobacterium paratuberculosis was used as reagent for an enzyme-linked immunosorbent assay (ELISA). This ELISA, which is endowed with species specificity with respect to the other mycobacteria, was applied to the analysis of bovine paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle caused by M. paratuberculosis. The distribution of anti-a362 antibodies in the cattle population was analyzed by a computer program (mixture population model) to determine a cutoff value for the test. The prevalence of a362 seropositivity in the Belgian bovine population was estimated to be 12%. The sensitivity of the a362 assay was 70%, as determined with reference sera from the U.S. National Repository of Paratuberculosis Specimens. Some 40% of the animals in the herds with paratuberculosis analyzed were found to be positive by the a362 assay. The latter proved to be 95% specific with respect to both healthy and tuberculous cattle.