Results of previous studies utilizing bioinformatic approaches in antigen-mining experiments revealed that secreted proteins are among the most frequently recognized antigens from Mycobacterium bovis. Thus, we hypothesized that the analysis of secreted proteins is likely to reveal additional immunogenic antigens that can be used to increase the specificity of diagnostic tests or be suitable vaccination candidates for mycobacterial infections. To test this hypothesis, 382 pools of overlapping peptides spanning 119 M. bovis secreted and potentially secreted proteins were screened for the ability to stimulate a gamma interferon response in vitro using whole blood from tuberculin-positive reactor (TB reactor) cattle. Of the 119 proteins screened, 70 (59%) induced positive responses in the TB reactor animals to various degrees. Strikingly, all but one of the 15 ESAT-6 proteins tested were recognized by at least 30% of the TB reactor animals, with 12 of the 22 most commonly recognized antigens belonging to this protein family. Further analysis of these data demonstrated that there was no significant difference in immunogenicity between the ESAT-6 proteins that were components of potentially intact ESX secretory systems and those corresponding to additional partial esx loci. Importantly for vaccine design, antigenic epitopes in some highly conserved regions shared by numerous ESAT-6 proteins were identified. However, despite this considerable homology, peptide-mapping experiments also revealed that immunodominant peptides were located in regions of amino acid variability.