Spin labeled phospholipid analogs were used to directly study changes in aminophospholipid translocase activity in activated platelets. In thrombin-activated platelets, the translocase activity was slightly stimulated, whereas no vesicle formation or proteolysis of cytoskeletal protein occurred. Ca2+ ionophore A23187-mediated activation produced vesiculation and proteolysis. Additionally, the translocase activity was completely inhibited, probably due to a sharp rise the intracellular Ca2+ concentration, as shown when platelets were activated in the presence of various A23187 and Ca2+ concentrations and by the recovery of the translocase activity when Ca2+ was complexed with EGTA. No translocase activity was found in vesicles. Whereas vesiculation and translocase inhibition can occur independently of proteolysis, this later accentuated the shedding phenomenon.