Mice were vaccinated with plasmid DNA (pDNA) encoding antigen 85A (Ag85A), Ag85B, or PstS-3 from Mycobacterium tuberculosis either in saline or formulated for intramuscular injections in VC1052:DPyPE (aminopropyl-dimethyl-myristoleyloxy-propanaminium bromide-diphytanoylphosphatidyl-ethanolamine) (Vaxfectin; Vical, Inc., San Diego, Calif.) or for intranasal instillations in GAP-DLRIE:DOPE (aminopropyl-dimethyl-bis-dodecyloxy-propanaminium bromide-dioleoylphosphatidyl-ethanolamine). These two novel cationic and neutral colipid formulations were previously reported to be effective adjuvants for pDNA-induced antibody responses. The levels of Ag85-specific total immunoglobulin G (IgG) and IgG isotypes were all increased 3- to 10-fold by formulation of pDNA in Vaxfectin. The level of production of splenic T-cell-derived Th1-type cytokines (interleukin-2 and gamma interferon) in response to purified Ag85 and to synthetic peptides spanning the entire Ag85A protein was also significantly higher in animals vaccinated with pDNA formulated in Vaxfectin. Cytolytic T-lymphocyte responses generated by pDNA encoding phosphate-binding protein PstS-3 in Vaxfectin were better sustained over time than were those generated by PstS-3 DNA in saline. Intranasal immunization with Ag85A DNA in saline was completely ineffective, whereas administration in GAP-DLRIE:DOPE induced a positive Th1-type cytokine response; however, the extent of the latter response was clearly lower than that obtained following intramuscular immunization with the same DNA dose. Combined intramuscular and intranasal administrations in cationic lipids resulted in stronger immune responses in the spleen and, more importantly, in the lungs as well. Finally, formulation in Vaxfectin increased the protective efficacy of the Ag85B DNA vaccine, as measured by reduced relative light unit counts and CFU counts in the spleen and lungs from mice challenged with bioluminescent M. tuberculosis H37Rv. These results may be of importance for future clinical use of DNA vaccines in humans.