Tumor promoters cause a variety of effects in cultured cells, at least some of which are thought to result from activation of the Ca2+-phospholipid-stimulated protein kinase C. One action of tumor promoters is the modulation of the binding and phosphorylation of the epidermal growth factor (EGF) receptor in A431 cells. To determine if these compounds act on the EGF receptor by substituting for the endogenous activator of C kinase, diacylglycerol, we compared the effects of the potent tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) with those of the synthetic diacylglycerol analog 1-oleyl 2-acetyl diglycerol (OADG). When A431 cells were treated with TPA, the subcellular distribution of C kinase activity shifted from a predominantly cytosolic location to a membrane-associated state; OADG also caused the disappearance of cytosolic C kinase activity. The shift in the subcellular distribution of C kinase, caused by TPA or OADG, correlated with changes in binding and phosphorylation of the EGF receptor. OADG, like TPA, caused loss of binding to an apparent high affinity class of receptors, blocked EGF-induced tyrosine phosphorylation of the EGF receptor, and stimulated phosphorylation of the EGF receptor at both serine and threonine residues. No difference between the phosphopeptide maps of receptors from cells treated with OADG or TPA was observed. Thus, it appears that tumor promoters can exert their effects on the EGF receptors by substituting for diacylglycerol, presumably by activating protein kinase C. Further, these results suggest that endogenously produced diacylglycerol may have a role in normal growth regulatory pathways.