We previously have reported that the lactic acid bacterium, Lactobacillus casei strain Shirota (LcS), shows marked antitumor activities and an ability to modify immune responses. In this study, we examined whether LcS can induce the production of interleukin (IL)-12 and interferon-gamma (IFN-gamma), which are important cytokines for antitumor and antimicrobial immunity, from murine splenocytes in vitro in order to clarify the mechanisms of its immune modification. Stimulation by LcS induced a marked production of IL-12 by X-ray-irradiated splenocytes (X-irr-Spl). The production of IL-12 by X-irr-Spl was independent of the presence of nylon wool column-passed splenocytes (NW-Spl). IFN-gamma was produced by splenocytes by the stimulation with concanavalin A (Con A). LcS showed a synergistic stimulatory effect on the ConA-induced production of IFN-gamma. In addition, X-irr-Spl were required for the IFN-gamma; production by NW-Spl treated with LcS. The IFN-gamma production was reduced by anti-IL-12 antibody treatment. NW-Spl produced IFN-gamma following treatment with recombinant IL-12. Thus, we confirmed that IFN-gamma production by splenocytes was the result of the production of IL-12 from X-irr-Spl stimulated by LcS. Furthermore, in BALB/c mice, the oral administration of viable LcS augmented the production of IFN-gamma but not that of IL-4 or IL-5 by splenocytes. Thus, we suggested that LcS primarily activated X-irr-Spl, probably macrophages, and these cells secreted IL-12. The IL-12 induced by LcS stimulated the production of IFN-gamma.