The mechanisms by which flow-imposed shear stress elevates intracellular Ca²⁺ in cultured endothelial cells (ECs) are not fully understood. Here we report finding that endogenously released ATP contributes to shear stress-induced Ca²⁺ responses. Application of flow of Hanks' balanced solution to human pulmonary artery ECs (HPAECs) elicited shear stress-dependent increases in Ca²⁺ concentrations. Chelation of extracellular Ca²⁺ with EGTA completely abolished the Ca²⁺ responses, whereas the phospholipase C inhibitor U-73122 or the Ca²⁺-ATPase inhibitor thapsigargin had no effect, which thereby indicates that the response was due to the influx of extracellular Ca²⁺. The Ca²⁺ influx was significantly suppressed by apyrase, which degrades ATP, or antisense oligonucleotide targeted to P2X₄ purinoceptors. A luciferase luminometric assay showed that shear stress induced dose-dependent release of ATP. When the ATP release was inhibited by the ATP synthase inhibitors angiostatin or oligomycin, the Ca²⁺ influx was markedly suppressed but was restored by removal of these inhibitors or addition of extracellular ATP. These results suggest that shear stress stimulates HPAECs to release ATP, which activates Ca²⁺ influx via P2X₄ receptors.