Release of FFA from membrane phospholipids was observed after incubation of umbilical cord vein-derived endothelial cells (HUVEC) with vascular endothelial growth factor (VEGF). In particular, we found an increase of arachidonate, stearate, and palmitate in a time-dependent manner with a peak at 30 min. The maximum increase was reached by arachidonate (4.4-fold), followed by stearate (2.2-fold) and palmitate (1.3-fold). The arachidonate increase can be ascribed to the activation of phospholipase A2 (PLA2). In fact, cells preincubated with arachidonyl trifluoromethyl ketone, a PLA2 inhibitor, showed a marked reduction in arachidonate mobilization. The role of Ca2+ in PLA2 activation was also investigated. Cells incubated with VEGF in the presence of EGTA showed a marked decrease in arachidonate mobilization, whereas incubation with the calcium ionophore A23187 alone produced an increase in arachidonate, although to a lesser extent compared with the VEGF stimulation. Incubation with A23187 in association with PMA produced the same increase in arachidonate as the VEGF treatment. Mitogen-activated protein kinase (MAPK) activity was also found to increase as a consequence of VEGF stimulation. Taken together, these results suggest that the VEGF-mediated activation of PLA2 in HUVEC is dependent on both MAPK-mediated phosphorylation and Ca2+ increase. Furthermore, the increase in stearate and palmitate likely is brought about by the activation of a pathway involving phospholipase D, phosphatidate phosphohydrolase (PAP), and DAG lipase. In fact, the increase in those FFA was prevented when HUVEC were stimulated with VEGF in the presence of ethanol (which inhibits the formation of phosphatidate), propranolol (a specific inhibitor of PAP), or RHC-80267 (a specific inhibitor of DAG lipase).