The Protein S Ac activity assay is based on the role of protein S as a cofactor to activated protein C (APC). Tests performed on three coagulation analyzers were studied to determine assay performance characteristics. The overall mean imprecision was 5.5%, 5.0% and 7.5% for within-run coefficient of variation (CV), run-to-run CV and total CV, resp. A total of 239 sample results yielded a correlation to the STA Protein S Clotting assay of r = 0.92 (slope = 0.96). A group of patients with APC resistance and addnl. underlying conditions, such as oral contraception, oral anticoagulation, pregnancy and recurrent thrombosis showed pathol. low protein S values with activity assays as well as with free antigen assays. To differentiate between the effects of APC resistance and other influencing factors, samples from healthy blood donors with APC resistance were measured with both assay types. Based on comparison of antigen and activity results, it can be concluded that APC resistance lowers protein S activity values by approx. 15% (rel.). No interference was observed from physiol. relevant concentration of factor V, factor VIII, triglycerides, Hb, bilirubin and heparin (low mol. weight heparin and unfractionated heparin). When samples are to be frozen, double centrifugation is the best procedure to reduce the platelet count. The stability of the sample was at least 4 h at room temperature, regardless of whether fresh or frozen samples were tested. Freezing had no observable effect on samples stored for up to 12 mo at -70°C.