Activated protein C (APC) resistance is usually associated with a single DNA mutation predicting replacement of Arg506 by Gln in factor V (FV). Studies using synthetic peptides suggest that FV residues 493-506 provide factor Xa (FXa) and protein S binding sites. Biochemical studies were performed to test the hypothesis that the Arg506Gln FV mutation causes APC resistance and to define the nature of the resistance of Gln506-FVa to APC. Purified Gln506-FV conveyed APC resistance to FV-deficient plasma in APTT and FXa-1-stage assays. Purified Gln506-FVa, generated either by thrombin or by FXa, was resistant to APC. Nonetheless, Gln506-FVa was not completely resistant to APC since it was inactivated by APC approximately 10-fold slower than normal Arg506-FVa, probably due to cleavage at Arg306. This reduced but significant susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance, especially for heterozygotes, is a relatively moderate risk factor for venous thrombosis. Cardiolipin promotes APC anticoagulant activity better than FXa coagulant activity, and antibodies from some antiphospholipid antibody syndrome patients downregulate APC activity. Thus, acquired APC resistance may contribute to pathogenesis of thrombosis in the antiphospholipid antibody syndrome.