The extrinsic coagulation pathway is initiated by the binding of plasma factor VII(a) (VIIa) to the cell surface receptor tissue factor (TF), which serves as the cofactor for the ligand protease VIIa in the activation of macromolecular substrate factors X and IX. The catalytic function of the TF.VIIa complex is regulated by a specific Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI), which forms a stoichiometric complex with the serine protease factor Xa (Xa), resulting in greatly accelerated inhibition of the extrinsic initiation complex as compared to free inhibitor. In the present study we identify specific residues in the TF-VIIa complex that are involved in the factor Xa-mediated acceleration of TFPI inhibitory function. VIIa residue Arg290, which contributes to extended recognition of macromolecular substrate factor X, is not involved in the interaction with the TFPI.Xa complex. In contrast, TF residues Lys165 and Lys166, which are important for the activation of factor X, are required for the accelerated inhibition of the TF.VIIa complex by TFPI mediated by factor Xa. These data indicate that similar interactions contribute to the assembly of substrate factor X as well as of product Xa after complex formation with TFPI, suggesting a central role for the carboxyl-terminal structural module of TF in regulating the proteolytic activity of TF.VIIa.