Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb), remains a major health problem. The only currently available vaccine, BCG, confers only variable protection and an improved vaccine is urgently needed. Administration of DNA vaccines encoding the secreted mycolyl-transferase Ag85A and the surface-exposed phosphate transport receptor PstS-3 elicit an immune response capable of protecting mice challenged with Mtb. In order to combine the protection against Mtb infection induced by these two DNA vaccines, we have cloned Ag85A and PstS-3 in pBudCE4.1 vector, in which antigenic expression is controlled by two independent promoters. (BALB/cxC57BL/6)F1 mice were vaccinated with this combination vaccine and immune responses were compared to those induced by vaccination with plasmids encoding the single antigens on pBudCE4.1 or pV1J.ns-tPA backbone. Antibody and Th1 type cytokine responses against Ag85A were comparable, whereas responses against PstS-3 were clearly lower in mice vaccinated with the combination plasmid, suggesting antigenic competition with the mycolyl-transferase being the dominant antigen.