This study combines two methodologies - vector expression of a genomic library and proteomics - to identify immunogenic proteins of Mycobacterium bovis. Immunization of BALB/c mice with a plasmid DNA pool from the library, containing approximately 8000 clones, induced a humoral response that facilitated the detection of 12 antigenic proteins by Western blotting. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry identified four proteins (Cpn60-1, HSP70, EF-Tu, and AdoHcyase). Such genomic immunization offers the possibility of in vivo screening of potential candidate M. bovis antigens.