The bacterium Burkholderia pseudomallei causes a life-threatening disease called melioidosis. In vivo experiments in mice have identified that a rapid IFN-γ response is essential for host survival. To identify the cellular sources of IFN-γ, spleen cells from uninfected mice were stimulated with B. pseudomallei in vitro and assayed by ELISA and flow cytometry. Costaining for intracellular IFN-γ vs cell surface markers demonstrated that NK cells and, more surprisingly, CD8+ T cells were the dominant sources of IFN-γ. IFN-γ+ NK cells were detectable after 5 h and IFN-γ+ CD8+ T cells within 15 h after addition of bacteria. IFN-γ production by both cell populations was inhibited by coincubation with neutralizing mAb to IL-12 or IL-18, while a mAb to TNF had much less effect. Three-color flow cytometry showed that IFN-γ-producing CD8+ T cells were of the CD44 high phenotype. The preferential activation of NK cells and CD8+ T cells, rather than CD4+ T cells, was also observed in response to Listeria monocytogenes or a combination of IL-12 and IL-18 both in vitro and in vivo. This rapid mechanism of CD8+ T cell activation may be an important component of innate immunity to intracellular pathogens.