Increasing evidence indicates that interaction of cells with fibronectin (Fn) affects many aspects of cellular responses including growth, morphology, differentiation, and activation. However, it is not known whether Fn could activate macrophages for the tumor cell killing. Here we report that Fn provides a signal for murine macrophage activation to tumoricidal activity. Tumoricidal activity was determined by the release of 51Cr from prelabeled P815 mastocytoma target cells. Fn alone had no effect, whereas recombinant interferon-gamma (rIFN-gamma) weakly induced C57BL/6 murine macrophages to kill P815 mastocytoma cells. However, combination of Fn with rIFN-gamma synergized to activate macrophages to lyse tumor cells in a dose dependent manner. Secretion of nitric oxide (NO) correlated with tumor cell killing, and the activated macrophages failed to kill tumor cell targets in the presence of NG-monomethyl-L-arginine (NGMMA), a competitive inhibitor of NO synthase (NOS). Fn, unlike lipopolysaccharide (LPS), alone had no effect on NO synthesis by itself and did not induce bioactive tumor necrosis factor-alpha (TNF-alpha) secretion from murine peritoneal macrophages. The data illustrate the potential for Fn to activate macrophage-mediated antitumor mechanisms in addition to its better characterized role as a cell adhesion molecule.