Activated protein C (APC) resistance is a major risk factor for venous thrombosis. Factor V (FV) gene mutations like FV(Leiden) (R506Q) and FV(R2) (H1299R) may cause APC resistance either by reducing the susceptibility of FVa to APC-mediated inactivation or by interfering with the cofactor activity of FV in APC-catalyzed FVIIIa inactivation. We quantified the APC cofactor activity expressed by FV(Leiden) and FV(R2) and determined the relative contributions of reduced susceptibility and impaired APC cofactor activity to the APC resistance associated with these mutations. Plasmas containing varying concentrations of normal FV, FV(Leiden), or FV(R2) were assayed with an APC resistance assay that specifically measures the APC cofactor activity of FV in FVIIIa inactivation, and with the activated partial thromboplastin time (aPTT)-based assay, which probes both the susceptibility and APC cofactor components. FV(R2) expressed 73% of the APC cofactor activity of normal FV, whereas FV(Leiden) exhibited no cofactor activity in FVIIIa inactivation. Poor susceptibility to APC and impaired APC cofactor activity contributed equally to FV(Leiden)-associated APC resistance, whereas FV(R2)-associated APC resistance was entirely due to the reduced APC cofactor activity of FV(R2). Thrombin generation assays confirmed the importance of the anticoagulant activity of FV and indicated that FV(Leiden) homozygotes are exposed to a higher thrombotic risk than heterozygotes because their plasma lacks normal FV acting as an anticoagulant protein.