After biosynthetic radiolabeling, polypeptides secreted by macrophages were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate and fluorography. Comparative studies of various macrophage populations (inflammatory macrophages, primed and activated macrophages, resident macrophages) showed that secretory activity is elaborately controlled according to macrophage developmental and functional state. When young macrophages activated to cytotoxicity against tumor cells by an appropriate eliciting agent and culture in the presence of lipopolysaccharide were compared to mature resident macrophages devoid of antitumor activity, the following changes were observed in the protein secretion phenotype: (1) New polypeptides are expressed: the intensity of four bands was increased more than five-fold on one-dimensional gels and 17 new spots were detected on two-dimensional gels. (2) The production of several major polypeptides is repressed: six bands were strongly decreased on one-dimensional gels, and 11 major spots (or groups of spots) were lost on two-dimensional gels. (3) The charge microheterogeneity pattern exhibited by several polypeptides on two-dimensional gels is shifted towards less acidic species.