Growth factor stimulation of quiescent cells induces a series of intracellular early and late events that ultimately lead to DNA synthesis and cell division. We describe here that production of phosphorylcholine is an essential component of the late events involved in the induction of DNA synthesis by platelet-derived growth factor (PDGF-BB), a prototype mitogen for fibroblasts. Moreover, phosphorylcholine itself is mitogenic when added exogenously to NIH3T3 cells, further indicating its role as a crucial intracellular messenger for DNA synthesis. Choline kinase, the first step in the route of phosphatidylcholine synthesis appears to be the critical regulatory enzyme in phosphorylcholine production, indicating that regulation of choline kinase represents a key step during mitogenic stimulation. We also describe that several growth factors (PDGF-AA, basic FGF, EGF and phorbol esters) rely on their ability to generate phosphorylcholine for their proliferating activity. In contrast, DNA synthesis induced by serum did not require phosphorylcholine. Moreover, the requirement for phosphorylcholine production in PDGF-stimulated cells can be over-ruled by addition of insulin. Thus, cell proliferation in NIH3T3 cells can be triggered off by alternative pathways and one of them involves generation of phosphorylcholine.