Murine macrophages from sites of inflammation develop toward tumoricidal competence by exposure to a macrophage-activating factor such as interferon-gamma (IFN-gamma). To explore the biochemical transductional events initiated by IFN-gamma, peritoneal macrophages from C57BL/6J mice elicited by various sterile irritants were treated in vitro with two pharmacologic agents that mimic the action of certain second messengers. Phorbol myristate acetate (PMA) and the ionophore A23187 cooperatively reproduced the ability of IFN-gamma to prime macrophages for tumoricidal function. Neither agent alone was able to prime macrophages. The two agents acted on the macrophages, and target susceptibility to kill was not altered by PMA and A23187. Only active phorbol esters, which are known to bind and stimulate protein kinase C, were able to cooperate with A23187 to induce priming. A cell-permeable synthetic diacylglycerol (sn-1,2-dioctanoyl glycerol) could also prime for cytolysis. In the presence of PMA, A23187, and EGTA, addition of Ca++ was sufficient for priming, whereas the addition of Mg++ was much less efficient. Priming by IFN-gamma, however, was not blocked by EGTA. Efflux of 45Ca++ from preloaded cells was significantly increased by A23187 and by IFN-gamma. Quin-2/AM, an intracellular chelator of Ca++, blocked priming by IFN-gamma. In summary, the data suggest that priming of macrophages for tumoricidal function by IFN-gamma involves, at least in part, alterations in protein kinase C and in levels of intracellular Ca++.