A search was initiated for M. paratuberculosis-specific proteins which may be useful for the diagnosis of Johne's disease. Protein expression profiles of 10 isolates of M. paratuberculosis, M. avium 18 (formerly M. paratuberculosis 18), and one isolate each of M. avium serotype 2, M. avium serotype 8, and M. bovis BCG were compared. SDS-PAGE analysis of [35S] methionine-labeled extracellular proteins revealed variability among the M. paratuberculosis isolates. SDS-PAGE analysis of [35S] methionine-labeled cellular proteins divided the M. paratuberculosis isolates into 2 groups based on a difference in the level of expression of a 28,000-dalton protein. Protein expression profiles of the M. paratuberculosis and M. avium isolates were similar. However, 2-dimensional gel analysis of [35S] methionine-labeled cellular proteins resolved 4 proteins, with molecular Masses of 28,000, 32,000-, 32,000-, and 42,000-daltons, which were expressed at a higher level in M. paratuberculosis than in M. avium. Two proteins, with molecular Masses of 43,000- and 60,000-daltons, were expressed at a higher level in M. avium than in M. paratuberculosis. Western (Immuno) blot analysis, using antiserum from a cow clinically infected with M. paratuberculosis as the primary antibody, suggested that the 42,000-dalton protein may be specific for M. paratuberculosis. A lambda gt11 expression library and a lambda DASH genomic library of M. paratuberculosis DNA were constructed to facilitate cloning of the gene encoding the 42,000-dalton protein and other genes encoding proteins with diagnostic potential.