DNA immunization is a promising new approach for the development of novel tuberculosis vaccines. In this study, the immune responses following the administration of single and combination tuberculosis DNA vaccines were evaluated. Single DNA vaccines encoding the MPT-63 and MPT-83 tuberculosis antigens evoked partial protection against an aerogenic challenge with M. tuberculosis Erdman in the mouse model of pulmonary tuberculosis. Immunization with a multivalent combination DNA vaccine (containing the ESAT-6, MPT-64, MPT-63, and KatG constructs) generated immune responses that indicated an absence of antigenic competition since antigen-specific cell-mediated and humoral responses were detected to each component of the mixture. More importantly, the combination vaccine elicited a strong protective response relative to the protection evoked by live BCG vaccine.