PURPOSE Reduction of the thrombogenicity of synthetic vascular grafts by endothelialization has been suggested. The purpose of this study was to examine some of the non-thrombogenic properties of cultured adult human great saphenous vein endothelial cells seeded on expanded polytetrafluoroethylene (ePTFE) grafts. METHODS Endothelialized grafts, control grafts, and wells were incubated with thrombin. Assays of thrombin loss from solution, thrombin coagulant activity, and protein C activation on the surface were obtained. The presence of thrombomodulin was confirmed with immunohistochemistry. RESULTS Significantly more thrombin was found on the ePTFE grafts or wells that underwent endothelialization, and larger amounts were lost from the thrombin solution compared with the control group. Thrombin enzyme activity on the endothelialization group was almost completely represented by activation of protein C and only to a minor extent by activity towards fibrinogen. CONCLUSIONS It is concluded that endothelial cells seeded on ePTFE retain the possibility to inhibit thrombin coagulant activity and to activate protein C. These findings provide support for the clinical applicability of cultured autologous endothelium on ePTFE grafts.