Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca²⁺]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca²⁺ indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca²⁺]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca²⁺]i) to green (high [Ca²⁺]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s⁻¹, no change in [Ca²⁺]i was observed during translocation of platelets, but [Ca²⁺]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca²⁺]i elevation. The intracellular Ca²⁺chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca²⁺]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca²⁺]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.