We investigated tumoricidal activation and induction of nitric oxide synthase (NOS) activity in macrophages from LPS-responsive (C3H/HeN) and -unresponsive mice (C3H/HeJ). Macrophages were incubated in vitro with a synthetic lipopeptide or with LPS and IFN-gamma. LPS and IFN-gamma activated C3H/HeN but not C3H/HeJ macrophages to lyse B16 melanoma cells. In contrast, lipopeptide and IFN-gamma activated macrophages from both strains of mice. Genistein, a specific inhibitor of protein tyrosine kinase, significantly blocked tumoricidal activation of macrophages from both strains of mice. Genistein did not affect tumor cell binding but significantly inhibited the production of nitric oxide. Genistein, herbimycin A, and tyrphostin inhibited the induction of NOS activity in macrophages from both strains of mice. These data suggest that protein tyrosine kinase activity is involved in the signal transduction pathway of LPS and other synthetic bacterial-related immunomodulators at a point preceding triggering of macrophage tumoricidal activation and expression of inducible NOS activity.