We investigated the effect of Lewis Lung carcinoma cells on the production of C3 by murine macrophages and examined the capacity of secreted C3 to opsonize Lewis Lung carcinoma cells. C3 released in culture from macrophages obtained from tumor-bearing C57Bl/6 mice as well as from normal macrophages exposed to Lewis Lung carcinoma cells in vitro was measured by hemolytic assays and by Western blot. We found that contact with tumor cells in vivo as well as in vitro enhanced the amount of C3 secreted by murine macrophages by a factor of 2-3. The inflammatory agent carrageenan caused only a small increase in the amount of secreted C3. On Western blots of concentrated macrophage supernatants, there was partial cleavage of secreted C3 which was, however, not more pronounced in the case of C3 from tumor-stimulated macrophages than from normal macrophages. Supernatants from normal as well as tumor-stimulated macrophages were capable of opsonizing Lewis Lung carcinoma cells as shown by their capacity to bind human erythrocyte in an immune adherence reaction. Pretreatment of the tumor cells with a protease inhibitor, PMSF, inhibited the capacity of the tumor cells to bind C3, suggesting that a tumor cell-associated protease might be involved in the binding of C3 to the tumor cell surface.