The kinetics of inhibition of prothrombinase during prothrombin conversion by antithrombin and antithrombin-heparin complexes was studied in a tubular flow reactor. Prothrombinase was assembled at a macroscopic phospholipid membrane, composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine, deposited on the inner wall of a glass capillary, by perfusion with a factor Xa-factor Va mixture. Measurement of thrombin production allowed estimation of the amount of prothrombinase present at the capillary wall. Perfusion with a mixture of prothrombin and antithrombin or antithrombin-heparin complexes caused a progressive decline of the prothrombinase activity. The rate of inactivation steeply decreased with increasing prothrombin concentrations, indicating competitive inhibition. Analysis of competitive inhibition data requires estimation of the time-dependent substrate concentration, Co, near the prothrombin converting surface using earlier developed transport theory [Billy, D., et al. (1995) J. Biol. Chem. 270, 1029-1034]. It appears that the inhibition rate is proportional to the fraction of enzyme, Km/(Km+Co), not occupied by substrate. The value of Km of prothrombinase estimated from the dependence of the inhibition rate on the prothrombin concentration (Km = 2-3 nM) is in excellent agreement with the value estimated from the substrate conversion rate (Km = 3 nM). Therefore inhibition of prothrombinase by antithrombin and antithrombin-heparin complexes is fully competitive with the substrate: prothrombin. Our results show that prothrombinase assembled on macroscopic lipid surfaces by virtue of its low Km value is protected for inhibition due to highly effective competition of prothrombin with antithrombin for the active site of factor Xa.