Radiosensitivity of various normal tissues is largely dependent on radiation-triggered signal transduction pathways. Radiation simultaneously initiates distinct signaling from both DNA damage and cell membrane. Specifically, DNA strand breaks initiate cell-cycle delay, strand-break repair or programmed cell death, whereas membrane-derived signaling through phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) enhances cell viability. Here, activation of cytosolic phospholipase A(2) (cPLA(2)) and production of the lipid second-messenger lysophosphatidylcholine were identified as initial events (within 2 min) required for radiation-induced activation of Akt and ERK1/2 in vascular endothelial cells. Inhibition of cPLA(2) significantly enhanced radiation-induced cytotoxicity due to an increased number of multinucleated giant cells and cell cycle-independent accumulation of cyclin B1 within 24-48 h of irradiation. Delayed programmed cell death was detected at 72-96 h after treatment. Endothelial functions were also affected by inhibition of cPLA(2) during irradiation resulting in attenuated cell migration and tubule formation. The role of cPLA(2) in the regulation of radiation-induced activation of Akt and ERK1/2 and cell viability was confirmed using human umbilical vein endothelial cells transfected with shRNA for cPLA(2)alpha and cultured embryonic fibroblasts from cPLA(2)alpha(-/-) mice. In summary, an immediate radiation-induced cPLA(2)-dependent signaling was identified that regulates cell viability and, therefore, represents one of the key regulators of radioresistance of vascular endothelial cells.