Tissue factor pathway inhibitor is a multivalent, Kunitz-type proteinase inhibitor. It directly inhibits factor Xa and, in a factor Xa-dependent fashion, produces feedback inhibition of the factor VIIa/tissue factor catalytic complex which is responsible for the initiation of coagulation. Human recombinant TFPI (rTFPI) produced in Escherichia coli was used to define the kinetic constants describing the human factor Xa:TFPI interaction. The inactivation of factor Xa by E. coli-rTFPI is indistinguishable from that of rTFPI produced in mammalian SK-hepatoma cells, suggesting that post-translational modifications such as glycosylation and phosphorylation do not play a major role in the inhibitory process. The slow, tight-binding inhibition of factor Xa follows the scheme: [formula: see text] Where the enzyme (E) and inhibitor (I) form an initial, immediate collision complex (EI) that then isomerizes slowly to a tightened final EI* complex. In the absence of other additions, the initial Ki (=k2/k1) and final Ki* for the inhibition of factor Xa by E. coli-rTFPI are 1.24 nM and 26.4 pM, respectively. In the presence of calcium ions (5 mM) the interaction between factor Xa and rTFPI is substantially weaker, with a Ki of 42.7 nM and Ki* of 85.2 pM. The addition of other components of the prothrombinase complex produces enhanced factor Xa inhibition predominantly through an effect on the initial Ki. In the presence of calcium ions and saturating concentrations of phospholipids and factor Va, the Ki and Ki* for factor Xa inactivation are 2.04 nM and 52.3 pM. The enhancing effect of heparin on the inhibitory process is concentration dependent and exhibits an optimum, reminiscent of the "template" model for heparin's acceleration of thrombin and factor IXa inhibition by antithrombin III. At optimal concentrations, the major mechanism of heparin action is also a reduction in the Ki of the initial encounter complex between factor Xa and rTFPI.