Serum from Mycobacterium bovis BCG-infected mice that had been challenged with lipopolysaccharide (LPS) exhibited a marked ability to induce gamma interferon (IFN-gamma) in cultures of spleen cells of normal mice in the presence of interleukin-2 (IL-2). The inducing activity became detectable in the circulatory system about 90 min after LPS challenge, disappeared at around 5 h, and was observable upon 640x dilution of the serum. Addition of monoclonal anti-IL-2 receptor antibody to the culture inhibited the induction by the serum. The activity induced high levels of IFN-gamma even in nylon wool-nonadherent cells, while concanavalin A failed to do so. Serum from uninfected mice challenged with LPS contained no such activity. The molecular weight of the active substance, estimated by gel filtration, was about 70,000. There were pronounced differences among mouse strains in the activities of the sera prepared, which paralleled the amounts of IFN-gamma produced in vivo. However, the levels of IFN-gamma produced in whole spleen cells and in nylon wool-nonadherent cells from mice of various strains were the same when stimulated with competent serum. These results indicate the existence of an unidentified factor that induces IFN-gamma in cooperation with IL-2 in macrophage-depleted splenocytes. They also suggest that IFN-gamma production in vivo is not genetically controlled at the lymphocyte level but, rather, at the level of synthesis of the unknown factor.