Cell proliferation is regulated by an appropriate combination of intracellular signals involving activation of kinases and the generation of phospholipid metabolites. We report here that growth factors induce a biphasic generation of phosphorylcholine (PCho) in quiescent NIH 3T3 cells, resulting in an early and transient increase at 100 s and a larger and sustained increase after 3 h of stimulation. Generation of PCho at both early and late times of growth factors stimulation results from the consecutive activation of phospholipase D (PLD) and choline kinase (ChoK). Production of PCho by specific growth factors seems an essential requirement for the early signals associated to activation of Raf-1 and MAP kinases, since blockage of choline kinase completely inhibited activation of Raf-1 and MAP kinases by PDGF or FGF. Both the transient early increase and the late sustained increase in PCho are required for the induction of DNA-synthesis, besides completion of the activation of the serine/threonine kinases cascade. Thus, our results strongly suggest that generation of PCho by the PLD/choline kinase pathway is one of the critical steps in regulating cell growth in NIH 3T3 stimulated by growth factors.