Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa dependent manner, inhibits the factor VIIa/tissue factor catalytic complex. The inhibitory effect of TFPI in prothrombin activation assays using purified components of the prothrombinase complex was examined. When factor Xa is added to mixtures containing TFPI, prothrombin, calcium ions, and nonactivated platelets or factor V and phospholipids, TFPI significantly reduces subsequent thrombin generation, and the inhibitory effect is enhanced by heparin. If factor Xa is preincubated with calcium ions and thrombin-activated platelets or factor Va and phospholipids to permit formation of prothrombinase before the addition of prothrombin and physiologic concentrations of TFPI (< 8 nmol/L), minimal inhibition of thrombin generation occurs, even in the presence of heparin. Thus, contrary to results in amidolytic assays with chromogenic substrates, prothrombinase is resistant to inhibition by TFPI in the presence of its physiological substrate, prothrombin. Higher concentrations of TFPI (approximately 100 nmol/L), similar to those used in animal studies testing for therapeutic actions of TFPI, do effectively block prothrombinase activity.