Continuous proteolysis resulting in consumption of major cytoskeletal proteins may be essential for platelet activation and aggregation. In this study we evaluated the effect of a known protease inhibitor, Leupeptin, on agonist induced platelet aggregation and secretion. Platelets exposed to 10 ugs/ml of Leupeptin did not aggregate in response to the action of thrombin (0.2 u/ml). However, a concentration of Leupeptin as high as 250 ugs/ml did not prevent arachidonate induced aggregation and secretion. Leupeptin (100 ugs/ml) effectively blocked thrombin (0.2 u/ml) induced elevation of cytosolic calcium, but did not affect arachidonate induced elevation of platelet intracellular calcium levels. At a concentration of 100 ug/ml, Leupeptin effectively blocked thrombin (0.5 u/ml) induced clot formation of platelet poor plasma, suggesting that it can exert its effect on thrombin by preventing fibrinogen degradation. Effective Ki for the competitive inhibition of thrombin induced hydrolysis of a chromogenic substrate, S2238, by Leupeptin was 2.4 uM. Leupeptin inhibition of platelet function was reversible by washing platelets free of the polypeptide. Results of our study demonstrate that Leupeptin inhibits thrombin induced platelet activation, probably by interfering with its proteolytic activity on the platelet surface membrane. However, inhibition of platelet surface membrane associated proteases did not prevent activation of platelets by other agonists.