Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants, and it has also been isolated and identified from patients with Crohn's disease, an inflammatory bowel disease. The control of Johne's disease has been hampered by the lack of a reliable diagnostic test because of the large degree of antigenic cross-reactivity between mycobacterial and non-mycobacterial species. To help identify specific antigen(s) or epitope(s), an M. paratuberculosis expression library was screened with antibodies and DNA probes. In total, 54 clones were randomly picked, purified, and characterized by DNA probes and monoclonal antibodies with known specificity to individual mycobacterial antigens. Four clones carrying the heat shock protein 65K-, two representing the secreted protein 32K-, three representing the 21K-, and 20 clones representing the specific insertion element of M. paratuberculosis (IS900)-encoding genes and their gene products were identified and characterized. Well-defined recombinant antigens and/or epitopes representing M. paratuberculosis may facilitate the development of specific diagnostic tests and the investigation of their role in these chronic diseases.