The role of platelet plasma membrane in mediating cellular response to external stimuli was investigated by studying phosphatidylinositol turnover and structural physical molecular alterations. Phosphoinositide turnover was evaluated by phosphatidylinositol breakdown and phosphatidic acid (PA) synthesis. The membrane structure was investigated by measuring fatty acid chain organization, flexibility and movements using the spin label method. When platelets are stimulated by thrombin, a rapid phosphatidylinositol bisphosphate (PIP2) breakdown is observed, accompanied by an immediate PA synthesis. At the same time a membrane-bound calcium liberation within the cell is revealed by the decrease in chlortetracycline fluorescence, and the dense granule constituent serotonin is released from the cell. These events result in disorganization of the platelet membrane as estimated by the decrease in order parameter. All these thrombin-induced effects occur in the presence of EDTA, thus being independent of external calcium and aggregation. By contrast ionophore A 23187 does not induce any PIP2 breakdown nor structural disorganization and the dense granule release measured at 10 seconds is only very weak. These results were confirmed by studies on pathological platelets in which PIP2 breakdown was normal when release was normal, such as in Glanzman thrombasthenia, or did not occur when no granule release was measurable as in Gray-platelet syndrome. It is suggested that membrane-bound calcium has a structural rather than a functional role.