The ability of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), to induce chemotaxis in three different populations of mouse peritoneal macrophages was studied. TPA in the range of 10(-9) to 10(-7) M produced a dose- and time-related increase in chemotaxis in resident, thioglycollate-elicited, and divinyl ether maleic anhydride copolymer-activated macrophages. A maximal response was obtained after 4 hr incubation with 10(-7) M TPA, and this concentration of TPA was as effective as inducing chemotaxis as was endotoxin-activated mouse serum. Orientation of macrophages towards TPA was also observed by microscopy. Within 2 hr, cells exposed to TPA sent out cytoplasmic processes along the TPA gradient. Parallel arrays of cells oriented towards the TPA were observed after 4 hr incubation. Two other diterpene tumor promoters, phorbol-12,13-didecanoate and mezerein, were also chemotactic for the macrophages, as was the peptide epidermal growth factor, which shares a number of effects with TPA on cells in culture. On the other hand, two phorbol esters inactive as tumor promoters, 4-alpha-phorbol-12,13-didecanoate and phorbol, were not chemotactic for macrophages. Retinoic acid, which inhibits tumor promotion, inhibited TPA-induced, but not endotoxin-activated mouse serum-induced chemotaxis. These findings, taken together with previous studies, indicate that phorbol ester tumor promoters are potent modulators of macrophage function.