The cytotoxic T-lymphocyte response (CTL) has been shown to be determinant in the clearance of many viral infections and hence, vaccine candidates against AIDS are designed to enhance this arm of the immune system. In this study, we have analyzed the antigen specific immune responses triggered in mice by different combinations of vaccine vehicles expressing the multiepitope polypeptide TAB13. This chimeric protein contains the V3 region of the gp120 from eight different HIV-1 isolates and was efficiently expressed by a DNA vector (DNA-TAB), and also by vaccinia virus recombinants (rVV) based either on the attenuated modified vaccinia virus Ankara (MVA-TAB) or Western Reserve (VV-TAB) strains. Inoculation of a DNA-TAB vector in priming followed by a booster with VV-TAB or MVA-TAB induces a humoral immune response against TAB13 protein and efficiently enhanced the CD8+ T cell response against V3 epitopes from HIV-1 isolates LR150, MN, and IIIB in comparison with animals immunized with two doses of DNA-TAB. A protocol that incorporates a DNA vector expressing IFN-gamma (DNA-IFN-gamma) with DNA-TAB in the priming, followed by a booster with MVA-TAB, triggered the highest values of specific CD8+ T cell response. By examining the cytokine pattern, the immune response induced by these vaccination approaches was predominantly of Th-1 type. These findings establish safe strategies for the enhanced generation of T cell mediated immunity to HIV-1 that can benefit in the design of an effective vaccine against AIDS.