Endothelial migration is one of the major events of pathological neovascularization. We compared the characteristics of Ca²⁺ mobilization in nonconfluent, confluent, and migrating endothelial cells. Migration of endothelial cells was induced by wounding the confluent cell monolayer. The basal intracellular Ca²⁺ concentration was lower in migrating cells and higher in confluent cells than in nonconfluent cells. Thapsigargin (TG)-induced Ca²⁺ leak and TG-evoked Ca²⁺ entry were accelerated in migrating cells, whereas the latter was suppressed in confluent cells. The ATP-induced Ca²⁺ transient was also much larger in migrating cells than in confluent cells. These alterations were also observed in a cell as an intracellular polarization, i.e., the leading edge showed an acceleration of TG-evoked Ca²⁺ entry and an augmentation of the ATP-induced Ca²⁺ transient. Endothelial migration was significantly suppressed by TG or cyclopiazonic acid. These observations suggest that the alterations of Ca²⁺ store site-related Ca²⁺ mobilizations, i.e., Ca²⁺ sequestration, release, and TG-evoked Ca²⁺ entry, may be involved in the cellular mechanisms of endothelial migration.