Listeria monocytogenes-specific, IFN-gamma-producing CD4+ T cells are induced in 5-day in vitro cultures of naive spleen cells with viable L. monocytogenes (VLM) but are not induced in cultures with heat killed L. monocytogenes (HKLM). Anti-IL-12 Abs abrogated the induction of the IFN-gamma-producing CD4+ T cells by VLM, suggesting an importance of IL-12 in the induction of IFN-gamma-producing CD4+ T cells. Accordingly, IL-12p40 was expressed by macrophages (M phi) from the cultures of whole spleen cells with VLM but not with HKLM, although HKLM induced IL-12p40 expression on M phi when nonadherent cells were removed before culture. In vivo analysis also showed that VLM induced IL-12p40 expression by M phi, whereas HKLM failed to induce it. On the other hand, the addition of anti-IL-10 mAb into cultures of whole spleen cells and HKLM resulted in an increase of IL-12p40 expression by M phi. Furthermore, we detected an expression of IFN-gamma by both adherent and nonadherent spleen cells at the early stage of stimulation with VLM before the appearance of IFN-gamma-producing CD4+ T cells. These data suggest that HKLM induce nonadherent spleen cells to produce IL-10 which may down-regulate IFN-gamma-producing CD4+ T cells by blocking IL-12 production by M phi. In contrast, VLM support IFN-gamma production by CD4+ T cells by stimulating M phi to produce IL-12 and IFN-gamma.