The proposal that epidermal growth factor (EGF) activates phospholipase D (PLD) by a mechanism(s) not involving phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis was examined in Swiss 3T3 fibroblasts. EGF, basic fibroblast growth factor (bFGF), bombesin, and platelet-derived growth factor (PDGF) activated PLD as measured by transphosphatidylation of butanol to phosphatidylbutanol. The increase in inositol phosphates induced by bFGF, EGF, or bombesin was significantly enhanced by Ro-31-8220, an inhibitor of protein kinase C (PKC), suggesting that PtdIns(4,5)P2-hydrolyzing phospholipase is coupled to the receptors for these agonists but that the response is down-regulated by PKC. Activation of PLD by EGF was inhibited dose dependently by the PKC inhibitors bis-indolylmaleimide and Ro-31-8220, which also inhibited the effects of bFGF, bombesin, and PDGF. Down-regulation of PKC by prolonged treatment with 4 beta-phorbol 12-myristate 13-acetate also abolished EGF- and PDGF-stimulated phosphatidylbutanol formation. EGF and bombesin induced biphasic translocations of PKC delta and epsilon to the membrane that were detectable at 15 s. In the presence of Ro-31-8220, translocation of PKC alpha became evident, and membrane association of the delta- and epsilon-isozymes was enhanced and/or sustained in response to the two agonists. The inhibitor also enhanced EGF-stimulated [3H]diacylglycerol formation in cells preincubated with [3H]arachidonic acid, which labeled predominantly phosphatidylinositol, but inhibited [3H]diacylglycerol production in cells preincubated with [3H]myristic acid, which labeled mainly phosphatidylcholine. These data support the conclusion that EGF can stimulate diacylglycerol formation from PtdIns(4,5)P2 and that PKC performs the dual role of down-regulating this response as well as mediating phosphatidylcholine hydrolysis. In summary, all of the results of the study indicate that PLD activation by EGF is downstream of PtdIns(4,5)P2-hydrolyzing phospholipase and is dependent upon subsequent PKC activation.