In earlier studies from this laboratory evidence was obtained for a physiological function of tissue factor pathway inhibitor (TFPI) as a regulator of hemostasis capable of preventing thrombotic complications that might otherwise result from exposure of blood to trace amounts of tissue factor (TF). However, it was not possible to conclude that the protective effect of TFPI stemmed solely from inhibition of factor VIIa/TF catalytic activity, since TFPI neutralizes stoichiometric amounts of factor Xa in forming an inhibited factor Xa/TFPI/factor VIIa/TF complex. Therefore, we examined the effects of immunodepletion of TFPI on the extent of coagulation initiated in rabbits by exposure to factor Xa and phospholipid in the absence of TF. In one experimental approach, factor Xa was generated endogenously with the factor X-activating fraction of Russell's viper venom (0.33 /xg/kg) in rabbits receiving an infusion of phosphatidylcholine/phosphatidylserine (PCPS) vesicles, 1 nig/kg over 2 hours. In a second approach, rabbits were injected with a complex of factor Xa (0.75 jug/kg) and PCPS (12.5 yug/kg). In contrast with the observed sensitization of TFPI-depleted rabbits to TF-induced coagulation, TFPI-depleted rabbits were not sensitized to coagulation initiated by factor Xa and phospholipid in the absence of TF. These data support the conclusion that the physiological function of TFPI in regulating TF-dependent coagulation stems primarily from its ability to inhibit factor VIIa/TF catalytic activity.