Protein S is a vitamin K-dependent protein cofactor to the anticoagulant, activated protein C (APC). This study examines the inhibition of human protein S anticoagulant activity by prothrombin. In the absence of protein S, the anticoagulant activity of APC measured in a Factor X(a), recalcification time, was comparable using normal or plasma adsorbed with Al(OH)3. Protein S was an effective cofactor to APC in Al(OH)3--adsorbed plasma, but was significantly less active in normal plasma. Analysis of the difference in the two plasmas revealed that normal plasma contained an inhibitor to the anticoagulant activity of protein S that was removed by Al(OH)3 adsorption. Purification of this inhibitory activity demonstrated that it was mediated by the vitamin K-dependent protein, prothrombin. Prothrombin purified by conventional techniques caused immediate, dose-dependent inhibition of the cofactor activity of protein S in the presence of phospholipids or platelets, but had no effect on the anticoagulant activity of APC. The inhibition was demonstrable using a Factor X(a) recalcification time, and studies of the rates of inactivation of purified Factor V(a). Increasing concentrations of protein S overcame the inhibition by prothrombin and kinetic analysis of the interaction demonstrated that prothrombin acted as a competitive inhibitor to protein S. Immunoabsorption of prothrombin from plasma using immobilized antiprothrombin antibodies was associated with the complete removal of the protein S inhibitory activity. We conclude that the anticoagulant activity of protein S is modulated by prothrombin and that this may represent another regulatory mechanism of the natural anticoagulant system.