Global screening of genomes and proteomes provides a powerful tool for identifying differences between two or more closely related organisms. In this study we have used comparative proteomics to identify proteins responsible for the phenotypic variations in the two genetically similar subspecies IS901⁺ Mycobacterium avium and Mycobacterium avium subspecies paratuberculosis. The advantage of this approach was that comparison of the proteomes of the two organisms would identify subspecies-specific proteins including the products of differential gene regulation that would not be detected by a comparative genomics approach. Comparisons were made between the proteomes of the two organismsgrown in vitro at both log and stationary phases of growth. Differentially expressed proteins were identified in both organisms and those found to be upregulated in M.a.paratuberculosis were further investigated by mass spectroscopy and Mascot analyses. The proteomes were compared from different strains of the organisms to ensure that the proteins identified were representative of the subspecies. Comparison with the in vivo proteome of M.a.paratuberculosis confirmed that the proteins were expressed during natural infection of the target species. The genes encoding the proteins of interest were cloned and expressed in Escherichia coli and the immunogenicity of the recombinant proteins determined to assess their potential as specific immunological reagents for diagnosis and epidemiological studies.