The third complement component, C3, is an important factor in the host defense mechanism in which monocytes/macrophages participate as the primary phagocytes. Monocytes/macrophages are the principal extrahepatic producers of C3, and this C3 production is thought to be regulated by several cytokines. In the present study, we demonstrated that human macrophage colony-stimulating factor (M-CSF) enhanced C3 production by human peripheral monocytes in serum-free culture. Analytical immunoblot and ELISA showed that the presence of M-CSF increased the production of intracellular pro-C3 and extracellular C3 for 24 h in a dose-dependent manner. To confirm the rapid effect of M-CSF on C3 production, we performed metabolic labeling of C3 with [35S]methionine. The production of [35S]C3 for the first 6 h in the presence of M-CSF was also increased as compared to that in the absence of M-CSF. In addition to the previously reported effects of M-CSF on monocytes/macrophages, such as the enhancement of C3 receptor expression and C3 receptor-mediated phagocytosis, we consider that the effects of M-CSF demonstrated in this study are of importance in the local immune system organization of C3 and monocytes/macrophages.