The dynamic change of cytoplasmic Ca2+ concentration ([Ca2+],) and morphological change were investigated simultaneously by confocal laser scanning microscopy using fluo-3 and by differential interference contrast optics in platelets activated by contact with the following types of surfaces: native glass and glass treated with poly-L-lysine (PLL), fibrinogen (Fg), or von Willebrand factor (vWF). The initial [Ca2+l, values just after the surface contact were comparable (approximately 100 nM) among platelets deposited on the four surface types. O n the PLL-surface, no morphological change or [Ca2+], elevation was observed. Glass-, Fg-, and vWF-surface adhered platelets showed pseudopod formation and spreading associated with the inhomogeneous [Ca2+], rise. The platelets on the Fg-surface were the most active in terms of [Ca2+], rise and morphological change. During pseudopod formation, the mean [Ca2+], value was maximal and localized high [Ca2+], zones were observed inside pseudopods, as well as in the center of the platelets. After spreading, high [Ca2+], zones still remained in the center of the cell. This new technique enabled simultaneous observation of [Ca2+], and cell shape and we clearly demonstrated a close relationship between [Ca2+], and morphological alterations.