A comparison was made of Mycobacterium paratuberculosis (M. ptb.; strain JTC303) protein expression and immunogenic antigens resulting from Watson-Reid culture filtrate (CF) vs. cellular extract (CE) preparations. Most proteins from CF had pI values of 4.0 to 5.5 whereas pI values for CE proteins had a broader range (4.0 to 7.0). Immunogenic proteins were more numerous from CF than CE preparations according to SDS-PAGE analysis with 31 sera collected from culture-confirmed cases of bovine paratuberculosis. This difference was even more evident after absorption of the sera with M. phlei antigens before immunoblotting. The most immunogenic CF proteins had molecular weights (MW) of 23 to 30 kDa. Twenty-four protein spots with the greatest staining intensity and different pIs and MWs were selected for analysis by MALDI-MS. Among these, five spots were successfully identified as ModD, PepA, ArgJ, CobT, antigen 85c complex and 9 additional hypothetical proteins were found. N-terminal peptide analysis of PepA, Antigen 85c, ModD, MAP1693c, MAP2168c, and MAP1022c revealed that each has 27 to 39 amino acids that may function as a signal sequence, supporting their being secreted through a Sec-dependent pathway. These results indicate that 14 secreted protein antigens found in a CF preparation of M. ptb. are strong candidates for use in improved serodiagnostic tests for paratuberculosis.