Negatively charged phospholipids accelerate blood coagulation; one suggestion is that the binding of the gamma-carboxyglutamic acid-containing zymogens to these lipid surfaces increases their effective concentration as substrates. Alternatively, the charged phospholipids could enhance the direct interaction of substrate with the catalytic complex, which is localized at the membrane surface. We distinguished the alternatives by using prothrombin fragment 1 to compete with the substrate for membrane binding sites without interfering with the direct enzyme-substrate interaction. In a tissue factor-factor VIIa system containing neutral phospholipids (to which the substrate does not bind), prothrombin fragment 1 had no significant effect on factor X activation (Km, 877 +/- 111 nM and 791 +/- 103 nM, with and without prothrombin fragment 1, respectively). In contrast, in a system containing 30% phosphatidylserine, prothrombin fragment 1 displaced phospholipid-bound factor X, increasing the free factor X concentration and the reaction velocity in all 22 determinations. As the velocity increases correlated only with the free factor X concentration, we conclude that the free factor X concentration controls this reaction velocity. The Km in the 30% phosphatidylserine system, calculated using free factor X concentration, was 41 +/- 5 nM and 63 +/- 9 nM, with and without prothrombin fragment 1, respectively. Thus, the negatively charged lipids decreased the intrinsic Km by over 90%. The methodology employed should be applicable to ligand-receptor systems in which ligand binds nonspecifically to the membrane surface.