Mitogenic stimulation of NIH3T3 fibroblasts with growth factors or ras oncogenes is associated with an increase in the levels of phosphorylcholine and diacylglycerol. Both metabolites could be generated as a result of direct activation of a phosphatidylcholine-specific phospholipase C (PC-PLC) or by a more complex pathway, involving activation of phospholipase D followed by choline kinase and phosphatidic acid-hydrolase. We show evidence indicating that the generation of phosphorylcholine and diacylglycerol follow independent mechanisms in both serum-treated and in ras-transformed NIH3T3 cells. No significant activation of a PC-PLC enzyme was observed. Instead, activation of a phosphatidylcholine-specific phospholipase D (PC-PLD) was detected. Moreover, while a fivefold constitutive activation of the endogenous PLD activity and a twofold increase on the levels of phosphatidic acid were observed in ras-transformed cells, very small alterations on these parameters were detected at late times after serum stimulation of quiescent cells. Thus, cell proliferation induced by ras oncogenes in fibroblasts cells may be functionally linked to activation of a PC-PLD enzyme. The differences found in the activation of this enzyme between ras-transformed and normal cells may constitute an important difference in mitogenic signalling between normal and transformed cells.