The inhibition of prothrombinase by tissue factor pathway inhibitor (TFPI) has been studied in the presence and absence of prothrombin, The rate constant of association of prothrombinase with full-length TFPI was 2.1 x 10(7) M(-1). s(-1) and 0.05 x 10(7) M(-1). s(-1) for the reaction with C-terminus truncated TFPI (TFPI1-161). The rate constant of dissociation was 0.65 x 10(-4) s(-1) in both cases. The rate constant of inhibition of prothrombinase by TFPI1-161 was similar to that of solution-phase factor Xa. In contrast, phospholipids and factor Va enhanced the association rate of the reaction between factor Xa and full-length TFPI by approx. 20-fold. Although TFPI, and in particular the full-length variant of the molecule, is a potent inhibitor of prothrombinase (overall inhibition constant of 3 pM), we also found that prothrombin competed very effectively with TFPI for the active site of factor Xa in the prothrombinase complex. A 50% reduction of the rate constant of inhibition was measured in the presence of 4 nM prothrombin, i.e. 0.2% of the plasma concentration of prothrombin. The physiological significance of TFPI as an inhibitor of prothrombinase activity is thus questionable.