Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed DNA vaccines encoding cytotoxic T lymphocytes (CTL) and T helper cell (Th) epitopes of the 38-kDa lipoglycoprotein of Mycobacterium tuberculosis and analyzed and compared their immunogenicities with that of pXJ38, a DNA vaccine encoding the entire 38-kDa protein (X. Zhu, N. Venkataprasad, H. S. Thangaraj, M. Hill, M. Singh, J. Ivanyi, and H. M. Vordermeier, J. Immunol. 158:5921–5926, 1997). Plasmid DNAs encoding a CTL epitope, P3 (pP3), a Th epitope (vTh), or both the Th and the P3 epitopes (pThP3) were prepared and tested in C57BL6/J (H-2b) mice. Our results confirmed that DNA immunization with pXJ38 induces strong CD8+ CTL and Th1 responses (high gamma interferon [IFN-γ], low interleukin-4 [IL-4]). Coadministration of plasmid DNAs encoding a Th epitope with those encoding a CTL epitope (vTh+pP3) elicited both antigen-specific CD8+ CTL and Th1 responses. High levels of IFN-γ were secreted by spleen cells from all plasmid DNA-vaccinated mice after in vitro stimulation with the recombinant 38-kDa protein. Small or undetectable amounts of IL-4 were observed, which indicates the induction of a Th1-like response. Multiple-epitope vaccination by vTh+pP3 or pThP3 resulted in a broader Th1 response to peptide or epitopes than the single-epitope plasmid DNAs. Antigen-specific immunoglobulin G2a was only detected in sera from mice immunized with the plasmid pXJ38, and not in mice immunized with the epitope-based DNA vaccines. Thus, the absence of an antibody response after immunization with epitope plasmid DNAs and their ability to trigger only a specific cellular immune response may prove to be important advantages for a vaccine against tuberculosis.