With the genome sequence available for Mycobacterium avium subspecies paratuberculosis (MAP), the technology is now developed for protein arrays to detect the presence of antibodies directed against MAP in host serum. The power of this approach is that it enables a direct comparison of MAP proteins to each other in relation to their immunostimulatory capabilities. In this study, over 60 MAP coding sequences were heterologously expressed and purified for use in a partial protein array. MAP proteins represented on this array include unknown hypothetical, cell surface, and MAP-specific proteins as well as previously characterized or known MAP antigens. The array was exposed to sera from MAP immunized rabbits and mice to identify immunodominant antigens in those artificial conditions. Furthermore, sera from non- and MAP-infected cattle were used to probe the array to identify antigens in the context of disease. Distinct sets of antigens emerged when data were compared between Johne's disease animals versus that of immunized mice and rabbits, indicating that immunodominant antigens in those artificial hosts may not represent antigens detected in the context of disease. Ten of the 64 proteins bound significant levels of antibody from clinical cattle. Of these ten proteins, three were previously identified as MAP antigens and the remaining seven represent novel antigens. Sera from three experimentally infected cattle identified at least one putative surface antigen detected early in infection. These data suggest this antigen may be useful in the early diagnosis of MAP infections. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of MAP.