The present study examines the role of antithrombin III (ATIII) and heparin in the inactivation of thrombin on rabbit aortae in vitro. Thrombin on the endothelium was functionally assayed with a synthetic chromogenic substrate (thrombins) or with fibrinogen (thrombinf), measured as the liberation of fibrinopeptide A. After incubation with thrombin, residual amounts were found on the endothelium. Surface-bound thrombinf constituted approximately one-half of surface-bound thrombins. Displacement of thrombin activity from the thrombin loaded endothelial surface was achieved with Polybrene. The thrombin could be quantitatively recovered in the Polybrene solution and equal amounts of thrombins and thrombinf were found. Incubation of endothelial segments with ATIII resulted in only a minimal uptake of ATIII on the surface but in a significantly increased capacity to inactivate thrombin, whereas incubation of the segments with heparin alone had the opposite effect. The effect of ATIII could be inhibited by prior incubation with Polybrene or by a subsequent incubation with heparin. It is concluded that the vessel wall content of ATIII is one determinant for the capacity to inhibit thrombin and that ATIII is bound to glycosaminoglycans on the vessel wall since it could be displaced by heparin.