The chimeric protein that relies on the T-cell epitopes of antigen 85B (Ag85B) and the 6-kDa early secreted antigen target (ESAT-6) has been demonstrated to augment the Th1 immune response. In this study, we developed a recombinant Mycobacterium bovis BCG (rBCG) strain that secretes the chimeric protein of Ag85B and ESAT-6 (rBCG-A(N)-E-A(C)). Immunization with this rBCG strain induced stronger antigen-specific gamma interferon (IFN-gamma) activities, as determined by an enzyme-linked immunospot assay, and higher levels of antigen-specific CD4(+) and CD8(+) T-cell responses than those in the control groups immunized with either rBCG expressing the Ag85B-ESAT-6 fusion protein (rBCG-A-E) or BCG. Likewise, rBCG-A(N)-E-A(C) significantly increased the level of production of the major Th1 cytokines IFN-gamma and tumor necrosis factor alpha in splenocyte cultures to levels comparable to those elicited by control BCG. Moreover, the antigen-specific immunoglobulin 2c (IgG2c)/IgG1 ratio for mice immunized with rBCG-A(N)-E-A(C) was also much higher than the ratios for the other immunized groups. Together, these results indicate that this rBCG-A(N)-E-A(C) strain enhances the Th1 cell-mediated response and may serve as a potential vaccine against M. tuberculosis.