In parallel to the behavior of many agents that activate complement via the alternative pathway and can stimulate macrophages to secrete lysosomal enzymes we investigated the interaction of mouse peritoneal macrophages cultured in a serum-free medium with various stimuli such as zymosan, polyanions, collagen type II, and immune complexes prepared from tetanus toxoid and pooled human anti-tetanus toxoid F(ab)2. All these stimuli induced the release of hydrolytic enzymes from macrophage in culture. The release was time and dose dependent and is not associated with loss of the cytoplasmic enzyme lactate dehydrogenase or any other sign of cell death. The mechanism of macrophage activation by these various agents is unknown. Macrophages have surface receptors for Fc and C3b with the capacity to bind immune complexes or C3b, respectively, and this is followed by activation of the cells. Activation via the Fc part can be excluded in these experiments. The possibility therefore arose that macrophages might be stimulated by endogenous C3 via the C3b receptor, since it is known that all the substances mentioned above can activate C3. To confirm this hypothesis we tried to inhibit this reaction by using an anti-C3-Fab preparation. There was hardly any detectably enzyme release after adding the anti-C3-Fab (dose dependent) together with the various stimuli to the macrophages. An unrelated Fab preparation showed no inhibitory effect. Furthermore, incubation of macrophages and the stimuli together with beta 1H and C3bINA abolished the effect to activate the macrophages. The observations now presented focus attention to the possibility that endogenous C3 could play a role in the stimulation of mouse peritoneal macrophages by various activators of the alternative pathway.