During agonist stimulation of endothelial cells, the sustained Ca²⁺ entry occurring through store-operated channels has been shown to significantly contribute to smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). However mechanisms linking Ca²⁺ stores depletion to the opening of such channels are still elusive. We have used Ca²⁺ and tension measurements in intact aortic strips to investigate the role of the Ca²⁺-independent isoform of phospholipase A₂ (iPLA₂) in endothelial store-operated Ca²⁺ entry and endothelium-dependent relaxation of smooth muscle. We provide evidence that iPLA₂ is involved in the activation of endothelial store-operated Ca²⁺ entry when Ca²⁺ stores are artificially depleted. We also show that the sustained store-operated Ca²⁺ entry occurring during physiological stimulation of endothelial cells with the circulating hormone ATP is due to iPLA₂ activation and significantly contributes to the amplitude and duration of ATP-induced endothelium-dependent relaxation. Consistently, both iPLA₂ metabolites arachidonic acid and lysophosphatidylcholine (LPC) were found to stimulate Ca²⁺ entry in native endothelial cells. However, only the later triggered endothelium-dependent relaxation through NO release, suggesting that LPC produced by iPLA₂ upon Ca²⁺ stores depletion may act as an intracellular messenger that stimulates store-operated Ca²⁺ entry and subsequent NO production in endothelial cells. Finally, we found that ACh-induced endothelium relaxation also depends on iPLA₂ activation, suggesting that the iPLA₂-dependent control of endothelial store-operated Ca²⁺ entry is a key physiological mechanism regulating arterial tone.