The tumor promoter phorbol myristate acetate (PMA) was compared to a lymphokine macrophage mitogenic factor (MMF) for its ability to induce replication of guinea pig peritoneal and alveolar macrophages. Like MMF, PMA induces DNA synthesis of both cell populations with peak thymidine incorporation at 72 hr of culture. Optimal concentrations of PMA for the peritoneal and alveolar cells were 1.6 x 10(-7) and 1.6 x 10(-9) M, respectively. The magnitude of the effect is slightly less than MMF but greater than that of phytohemagglutinin or concanavalin A. Indomethacin added to inhibit prostaglandin synthesis potentiates the effects of MMF but has little effect on the actions of PMA and the other mitogens. Potentiation by indomethacin of the effects of PMA on the peritoneal cell was observed only at the suboptimal concentration of PMA (1.6 x 10(-8) M). By adherence criteria and density gradient fractionation, the cell responding to PMA is confirmed to be the macrophage. Cell counts and nuclear radioautography confirm that replication in this system is reasonably well reflected by thymidine incorporation. The effects of PMA and its analogs as macrophage mitogens correlate with their tumor-promoting effects. Both PMA and MMF induce early increases in peritoneal macrophage levels of cyclic 3':5'-guanosine monophosphate without changes in the levels of cycles 3':5'-adenosine monophosphate. These studies indicate that PMA offers a useful probe of macrophage function.