Interferon-gamma (IFN-gamma) activation of macrophages is a crucial step in the early innate defence against bacterial infection. This innate IFN-gamma is thought to be produced mainly by natural killer (NK) cells through activation with interleukin (IL)-12p70 secreted by macrophages and dendritic cells (DCs) that have sensed bacterial products. However, a number of reports have shown that bacterial stimuli are unable to induce macrophages and/or DCs to produce sufficient amounts of IL-12p70 unless these cells are primed by IFN-gamma. It remains, therefore, unsettled how initial IFN-gamma is produced. In a previous study, we reported a novel IFN-gamma production pathway that was associated with cell death in macrophages caused by intracellular bacteria like Listeria monocytogenes (LM) and Shigella flexneri. In this study, we showed that cell death of bone-marrow-derived macrophage (BMM) cells following in vitro infection with Staphylococcus aureus (SA), an extracellular bacterium, can also stimulate this IFN-gamma production pathway. We also unequivocally demonstrated by using BMM cells from IL-12-deficient mice that the bacterial-infected macrophage cell death-mediated IFN-gamma production can occur without IL-12 although the magnitude of the response is much smaller than that in the presence of IL-12. The enhancing effect of IL-12 on this response proved to be attributable to the negligible amounts (0.5 approximately 1.5 pg/ml) of IL-12p70 but not to the large amounts of IL-12p40 that were both secreted by SA- and LM-infected macrophages. Taken all together, we propose that macrophage cell death caused by bacteria may trigger the initial IFN-gamma production at an early stage of bacterial infection.