Intramuscular injection of bacterially derived plasmid DNA results in the development of both humoral and cellular immune responses against plasmid-encoded antigens. Immunostimulatory CpG sequences within bacterial DNA are thought to enhance this process by stimulating the secretion of proinflammatory cytokines such as interferon γ (IFNγ) by cells of the innate immune system. Although IFNγ induction by CpG elements within plasmid DNA has been documented in vitro and more recently in vivo, and coimmunization with plasmids expressing IFNγ has been shown to enhance DNA-immunization-induced immune responses, it is unclear if IFNγ is necessary for successful DNA immunization. To address this issue, we compared humoral and cellular immune responses in wild-type and IFNγ-deficient mice vaccinated with a plasmid (pCMVNP) expressing the nucleoprotein gene from the arenavirus lymphocytic choriomeningitis virus (LCMV). IFNγ-positive (BALB/c) and IFNγ-negative (GKO) mice responded to DNA vaccination by the development of antigen-specific CD8⁺ T cells, which were detectable directly ex vivo by intracellular cytokine staining and comprised 0.7–2.5% of all CD8⁺ T cells in the vaccinee. DNA vaccines also induced virus-specific cytotoxic T lymphocytes (CTL), even in the absence of IFNγ. DNA vaccination of both mouse strains also was associated with a significant reduction in viral titers after LCMV challenge, indicating that, at least in the presence of other immune effector mechanisms, IFNγ is not required for induction of protective anti-viral immunity by DNA immunization. No quantitative differences were observed in antiviral IgG levels among GKO and BALB/c vaccinees, although GKO mice did exhibit a significant reduction of the IgG2a:IgG1 ratio, in agreement with the previously documented requirement for IFNγ in isotype switching to IgG2a. Immunized BALB/c mice produced similar levels of both IgG1 and IgG2a, indicating a mixed Th1/Th2 response to intramuscular immunization with pCMVNP. These results show that IFNγ induction by bacterially derived plasmid DNA does not contribute to the magnitude of the antibody response and is not required for the induction or short-term maintenance of DNA-induced CTL. However, IFNγ is necessary for the development of IgG2a antibodies that may be crucial for protection against some pathogens.