IL-12 is a potent proinflammatory cytokine. The effects of IL-12 are thought to be mediated by IFN-gamma production by NK, NKT, and T cells. In this study, we show that although IL-12 stimulates NK and NK1.1(+) T cells in bulk mouse splenocytes, it does not significantly stimulate purified NK cells, indicating that other cells are required. IL-12 stimulates T cell-deficient spleen cells and those depleted of macrophages. Unexpectedly, the depletion of dendritic cells also has little effect on the stimulation of spleen cells with IL-12. In contrast, B cell depletion almost completely inhibits IL-12-induced IFN-gamma production and B cell-deficient spleen cells are poorly stimulated with IL-12. Furthermore, purified NK cells are stimulated with IL-12 in the presence of purified B cells. Thus, B cells are necessary and also sufficient for the stimulation of purified NK cells with IL-12. Whereas spleen cells from IL-18-deficient mice are not stimulated with IL-12, NK cells purified from IL-18-deficient mice are stimulated with IL-12 in the presence of wild-type (WT) B cells, and WT NK cells are not stimulated with IL-12 in the presence of IL-18-deficient B cells. Cell contact between B and NK cells is also required for IL-12-induced IFN-gamma production. Finally, B cell-deficient mice injected with IL-12 produce significantly less IFN-gamma and IL-18 in the sera than WT mice do. Thus, stimulation of NK cells with IL-12 requires B cell cooperation in vitro as well as in vivo.