The eradication of the economically important disease, Johne's disease, has been hampered by the lack of simple accurate diagnostic tests. We have begun to dissect the antigenic structure of M. paratuberculosis to identify the species-specific antigen(s) required for development of a sensitive and specific diagnostic test and to understand the role of individual proteins in the pathogenesis of Johne's disease and possible Crohn's disease. An M. paratuberculosis (strain linda) expression library was constructed in a phagmid vector and screened with adsorbed hyperimmune rabbit anti-M. paratuberculosis serum and pooled polymerase chain reaction (PCR)-DNA product probes representing 65K and 32K-encoding genes and the IS900 of M. paratuberculosis. The derived recombinants contained 3 to 5 mycobacterial genome equivalents; 60% of recombinants contained inserts with the mean size of 2.8Kb. Three recombinants from 116 to 65 independent clones reacted with antibodies and pooled PCR-DNA probes, respectively, reacted with both antisera and PCR-DNA product probes. 25 of 29 clones selected by the pooled PCR probes reacted with the IS900 PCR-probe; 4 clones including the expressed one hybridized with 32K and 65K PCR DNA probes. Because 65K and 32K antigens are known to be major stimulants of cellular and humoral immunity against mycobacteria, our 4 putative species-specific clones are under investigation to identify species-specific epitope(s) useful in the development of rapid diagnostic tests as well as in the investigation of their role in pathogenesis of disease.