Bloodcoagulation is triggeredby the formationof acomplex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF–FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa–TFPI exerts feedback inhibition of TF–FVIIa. The FXa-depend-ent inhibition of TF–FVIIa activity by TFPI leads to for-mation of the quaternary complex TF–FVIIa–FXa–TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF–FVIIa–FXa–TFPI. .