Thrombin activity and inhibition were assayed on the aortic surface of dogs and pigs. After sacrifice, the aortae were excised and thrombin activity was measured with an amidolytic assay. A small thrombin activity was found on the endothelium. Heparinization of the animals lowered endothelial thrombin activity. After exposure of the aortic endothelium to a thrombin/albumin solution in vitro, thrombin activity disappeared from the solution and was recovered on the surface. De-endothelialized vessels took up more thrombin than those with intact endothelium. Endothelium confined thrombin was rapidly inactivated when exposed to plasma. A slow inactivation was seen upon exposure to a modified Ringer's solution. Thrombin confined to de-endothelialized aortae, i.e. to medial structures, was always inactivated at a slow rate irrespective as to whether it was exposed to plasma or Ringer's solution. It is concluded that endothelium and subendothelium bind thrombin and subsequently inactivate it. The inactivation proceeds faster on endothelium when exposed to plasma. The possible role of glycosaminoglycans is discussed.