The rate of inactivation of activated Factor V (Factor Va) by activated protein C can be enhanced by the addition of plasma. Plasma alone, however, had no effect upon Factor Va activity. This observation indicated that plasma may contain a cofactor for activated protein C. The cofactor activity was removed from plasma by barium citrate absorption and was eluted from the barium pellet. The cofactor activity eluted slightly ahead of prothrombin when chromatographed on quaternary aminoethyl (QAE)-Sephadex. The activity appeared to co-chromatograph with a protein that has been designated protein S. Purified cofactor-protein S had no effect upon the activity of purified bovine Factor Va either in the presence or in the absence of phospholipid. Purified cofactor-protein S caused a large enhancement in the rate of inactivation of Factor Va by activated protein C in the presence of phospholipid. These results indicate that protein S may be a cofactor for activated protein C.