—The endothelial α_vβ₃ integrin occurs luminally, where its ligation by soluble agents may induce inflammatory signaling. We tested this hypothesis in bovine pulmonary artery endothelial cell monolayers with the use of vitronectin and cross-linking antibodies to ligate and aggregate the integrin. We quantified the endothelial cytosolic Ca²⁺ concentration ([Ca²⁺]_i) according to the Fura 2 ratio imaging method in single cells of confluent monolayers. At baseline, endothelial [Ca²⁺]_i levels remained steady at 86 nmol/L for >20 minutes. Cross-linking of the α_vβ₃ integrin through the sequential exposure of monolayers to anti-α_vβ₃ monoclonal antibody LM609 and secondary IgG resulted in a [Ca²⁺]_i increase of 100% above baseline. This increase commenced in <0.5 minute, peaked in <2 minutes, and decayed to baseline in ≈5 minutes. Similar responses occurred after the addition of vitronectin (400 μg/mL). In contrast, external Ca²⁺ depletion blunted the cross-linking–induced [Ca²⁺]_i increase by 60%, a response that was completely inhibited when the monolayers were also pretreated with thapsigargin. Thus, the [Ca²⁺]_i increase was attributable in part to the release of Ca²⁺ from endosomal stores but mostly to Ca²⁺ influx across the plasma membrane. Induced aggregation of the α_vβ₃ integrin enhanced tyrosine phosphorylation of phospholipase C-γ1 and increased the accumulation of inositol-1,4,5-trisphosphate. Genistein, a broad-spectrum tyrosine kinase inhibitor, abrogated both of these effects, as well as the α_vβ₃-induced [Ca²⁺]_i increases. We conclude that aggregation of the endothelial α_vβ₃ integrin induces a rapid tyrosine phosphorylation–dependent increase in [Ca²⁺]_i. This response may subserve the inflammatory role of α_vβ₃ integrin in blood vessels.