The inhibition of thrombin as well as of factor Xa has been thought to occur primarily in plasma through the neutralizing action of the serine protease inhibitor antithrombin III (AT-III). However, inhibition of thrombin and Xa by this mechanism may not be sufficient for effective elimination of these clotting factors in states of increased coagulation activity. The potential role of the vascular endothelium in the inhibition of clotting factor activities has therefore received attention in recent years. The aim of the present investigation was to characterize the binding and inhibition of thrombin and factor Xa to the vascular endothelial cell (EC), smooth muscle cell (SMC) and rat hepatoma cell (RHC) in vitro, as well as to evaluate the effects of plasma constituents upon the inhibition of these factors. Purified bovine thrombin and factor Xa were used. The enzymatic activities of both factors were assayed using chromogenic substrates. The cells were exposed for 5 U/ml thrombin or 0.5 U/ml factor Xa. After 10 minutes incubation, the initial thrombin activity in the solution had decreased by about 20% in case of EC and SMC and about 11% when incubated with RHC. Thrombin activity recovered from the cell surface amounted to 0.02 U/cm2. When the cells with the surface bound enzyme were incubated with defibrinogenated plasma or AT-III for 30 seconds, only about 10% and 25-40%, respectively, of initial activity could be found. In similar experiments with factor Xa, after 10 minutes incubation, the initial activity in the solution had decreased by 10%. Factor Xa activity recovered from the cell surface was 0.001 U/cm2. After 30 seconds exposure to AT-III, no cell surface related factor Xa activity was recovered, whereas 10% of the cell surface activity was recovered after incubation with defibrinogenated plasma. It is concluded that thrombin and factor Xa are taken up and inhibited by EC, SMC and RHC cell surfaces in similar ratios suggesting that cell surface-mediated inactivation of activated clotting factors is not restricted to vascular wall cells. The inactivation of factor Xa was dependent on AT-III, however, the inactivation of thrombin was further promoted by an additional unidentified plasma constituent