Recently, our laboratory described a defect in anticoagulant response to activated protein C (APC). This response, APC resistance, was shown to be inherited and associated with familial thrombophilia. As other possible mechanisms were excluded, APC resistance was hypothesized to be due to deficiency of a previously unrecognized cofactor of APC. The aim of the present study was to isolate and characterize this factor. Plasma from an individual with pronounced inherited APC resistance was used as test plasma in a biological assay which monitored APC cofactor activity during its isolation from normal plasma. A purification procedure was devised that yielded a protein which was shown to be identical to coagulation factor V. It proved impossible to separate the APC cofactor activity from factor V, even by affinity chromatography using a monoclonal antibody against factor V. The affinity-purified factor V corrected the poor anticoagulant response to APC of APC-resistant plasma in a dose-dependent manner. Because the APC-resistant plasma contained normal levels of factor V procoagulant activity, the results indicated APC resistance to be due to a selective defect in the anticoagulant function of factor V. The present results show factor V not only to express procoagulant properties after its activation by thrombin but also to play an important part in the anticoagulant system as cofactor to APC.