Changes in intracellular calcium concentration ([Ca2+](i)) and focal adhesion sites of cultured bovine aortic endothelial cells (BAECs) were simultaneously visualized in real time. Local [Ca2+](i) transients were observed at the rear edges of spontaneously migrating BAECs. Furthermore, the majority of starting regions of [Ca2+](i) transients retracted continuously. Frequency of [Ca2+](i) transients increased with the application of fluid flow. The majority of starting regions of flow-induced [Ca2+](i) transients retracted following the occurrence of [Ca2+](i) transients. In addition, retracted areas were distributed in the upstream regions of the cell. Application of GdCl3, a mechanosensitive cation channel blocker, resulted in a clear reduction of [Ca2+](i) transients and rear retractions in cases of spontaneous and flow-induced BAEC migration. Flow-induced directional rear retractions were also inhibited. Consequently, we conclude that local [Ca2+](i) transients play an important role in the migration of BAECs with respect to rear retraction. Furthermore, how-induced [Ca2+]; transients regulate directional rear retraction under flow conditions.