Lipid-derived metabolites play an important role in the regulation of cell responses to external stimuli, including cell growth control, transformation and apoptosis. Phospholipase D (PLD) is one of the critical elements in the regulation of lipid metabolism and the generation of second messengers, some of them involved in cell growth control. Oncogenic Ras proteins affect the activity of PLD by two alternate mechanisms, involving a positive activation and a feedback negative loop. Here we investigate the involvement of the proto-oncogenic Ras protein in the physiological activation of PLD induced by platelet-derived growth factor (PDGF). Over-expression of the wild type Ras protein or some of its regulatory components, such as Shc or Grb2, induces an amplification of PLD activation by PDGF challenge. Furthermore, blocking the endogenous Ras by expression of the dominant negative mutant, H-Ras-Asn17 completely eliminated the activation of PLD by PDGF. Thus, PDGF requires a complex system for PLD regulation implying the existence of at least two positive regulatory pathways, a Ras-dependent and a PKC-dependent mechanism. These results imply that PLD is an important element in signaling by Ras proteins that is altered after ras-induced transformation.