Interleukin-12 (IL-12) is a critical cytokine that affects many of the biological functions of NK cells and T cells. We have previously shown that both human and murine NK cells are important in host defense against Mycobacterium avium complex and act by secreting cytokines that induce macrophages to inhibit the growth of intracellular M. avium. To define the role of IL-12 in M. avium complex infection, we stimulated human NK cells with recombinant human IL-12 at 0.01 to 1 ng/ml for 24 h and used the tissue culture supernatant to treat human monocyte-derived macrophage monolayers infected with M. avium. IL-12 had no direct effect on M. avium-infected macrophages, but culture supernatant from IL-12-treated NK cells activated macrophages to inhibit the growth of intracellular M. avium in a dose-dependent manner. Stimulation of NK cells with IL-12 in combination with tumor necrosis factor alpha (TNF-alpha) or IL-1 increased the ability of supernatant from NK-cell culture to limit M. avium growth within macrophages, compared with that of culture supernatant from IL-12-treated NK cells. Results with supernatant from nonstimulated NK cells were similar to those with supernatant from untreated controls. Treatment of supernatant from IL-12-stimulated NK cells with anti-TNF-alpha, anti-granulocyte-macrophage colony-stimulating factor, but not anti-gamma interferon antibodies decreased the ability of NK-cell supernatant to induce anti-M. avium activity in infected macrophages. Treatment of macrophage monolayers with anti-transforming growth factor beta antibody before adding supernatant from IL-12-stimulated NK cells was associated with an increase of anti-M. avium activity compared with that of supernatant from IL-12-treated NK cells. These results suggest that IL-12 has a role in host defense against M. avium and that the effect of IL-12 is dependent chiefly on TNF-alpha and granulocyte-macrophage colony-stimulating factor.