The facultative intracellular bacterium Francisella tularensis is capable of causing systemic infections in various hosts, including mice and humans. The liver is a major secondary site of F. tularensis infection, but hepatic immune responses to the pathogen remain poorly defined. Immune protection against the pathogen is thought to depend on the cytokine gamma interferon (IFN-γ), but the cellular basis for this response has not been characterized. Here we report that natural killer cells from the livers of naïve uninfected mice produced IFN-γ when challenged with live bacteria in vitro and that the responses were greatly increased by coactivation of the cells with either recombinant interleukin-12 (IL-12) or IL-18. Moreover, the two cytokines had strong synergistic effects on IFN-γ induction. Neutralizing antibodies to either IL-12 or IL-18 inhibited IFN-γ production in vitro, and mice deficient in the p35 subunit of IL-12 failed to show IFN-γ responses to bacterial challenge either in vitro or in vivo. Clinical isolates of highly virulent type A Francisella tularensis subsp. tularensis organisms were comparable to the live attenuated vaccine strain of Francisella tularensis subsp. holarctica in their ability to induce IL-12 and IFN-γ expression. These findings demonstrate that cells capable of mounting IFN-γ responses to F. tularensis are resident within the livers of uninfected mice and depend on coactivation by IL-12 and IL-18 for optimum responses.