Three proteins secreted from Mycobacterium tuberculosis, Ag85B, ESAT-6 and MPT63 were selected as antigens for making combined DNA vaccine by immunizing mice. The immune response induced by the vaccine and its protective efficacy were studied. It was demonstrated that when mice were immunized with the combined DNA vaccine, the titer of antibody for Ag85B in serum increased to more than 10(5), but the titers of ESAT-6 and MPT63 specific antibodies were undetectable. After the final immunization, the level of gamma specific for Ag85B, ESAT-6 and MPT63 reached (17.0+/-7.0) u/ml, (6.0+/-0.8) u/ml and (11.9+/-8.0) u/ml, respectively. Mice, that were inoculated with the empty eukaryotic expression vector pJW4303 DNA, produced negligible amounts of antigen-specific INF-gamma. The combined DNA vaccine resulted also in significantly reduced amount of bacteria in the lungs of experimental mice. Microphotographs showed clearly that these lungs were better protected against Mycobacterium tuberculosis challenge than control mice. The combined DNA vaccine reported in this work shed new lights on the prophylactic protection against tuberculosis.