Abstract Thrombin binds to and is inactivated by the endothelium. The inactivation is potentiated by plasma. The present investigation was designed to clarify the role of vessel wall glycosaminoglycans (GAG) and plasma antithrombin III (AT III) in the inactivation and binding of thrombin by endothelium. Thrombin was shown to bind to vascular endothelium and artificial surfaces containing GAG:s. The binding could be inhibited on both types of surfaces by pretreating them with protamine. Thrombin bound to endothelium was rapidly inactivated in the presence of plasma but only slowly if the plasma was replaced by AT III, AT III-depleted plasma or a balanced salt solution. It is concluded that thrombin binds to vessel wall GAG:s and is inactivated by the endothelium. Potentiation of the inhibition of the endothelially bound thrombin by plasma is dependent upon presence of AT III but an additional plasma factor is also required.