In this paper we describe the effect of activated protein C (APC) on thrombin generation initiated in platelet-poor plasma via the extrinsic or the intrinsic pathway. Thrombin was determined with a specific chromogenic substrate and quantitated by calculating the time integral of the thrombin generation curve, i.e. the endogenous thrombin potential (ETP). Addition of APC to normal plasma after both extrinsic and intrinsic initiation of coagulation resulted in a dose-dependent inhibition of thrombin generation as reflected by the decrease in ETP. Data obtained in intrinsically triggered plasma of normal individuals were subject to large variation. Therefore, the effect of APC on thrombin generation in APC-resistant plasmas was only studied in extrinsically stimulated reaction systems. APC had much less effect on the ETP of plasma from individuals that were heterozygous or homozygous for the mutation Arg506→ Gln506in factor V (APC resistance). There appears to be a linear relationship between the ETP and the amount of α2-macro-globulin-thrombin complex (α2M-IIa) that accumulates in plasma during thrombin formation. Since the α2M-IIa complex possesses amidolytic activity, we measured the effect of APC on thrombin generation via the so-called normalized APC sensitivity ratio (APC-sr). The latter was defined as the ratio of the end levels of amidolytic activity of the α2M-IIa complex determined in the presence and absence of 50 nM APC (α2M-IIa+APC/(α2M-IIa-APC) divided by the ratio of a normal plasma pool. Significant differences (P< 0.001) were observed between APC-sr of plasmas from normal individuals (APC-sr: 0.5–1.9,n= 25) and of plasmas from individuals that were heterozygous (APC-sr: 2.1–6.7,n= 17) or homozygous APC resistant (APC-sr: 3.9–5.9,n= 5). There was no overlap between APC-sr of normal plasmas and plasma from individuals, bearing the factor V mutation. Abnormal APC-sr in certain plasmas (pregnancy, use of oral contraceptives, anticoagulant therapy, protein S deficiency or lupus anticoagulant) were corrected by performing the assay on a plasma sample that was diluted 10-fold in factor V-deficient plasma. Our data show that measurement of the effect of APC on the ETP yields valuable information about the (pro)thrombotic status of plasma (e.g. APC resistance, pregnancy, use of oral contraceptives).