The heparin-enhanced antithrombin III/thrombin reaction was studied under experimental conditions where the dependence of the reaction velocity on the concentrations of thrombin and antithrombin III could be determined. The results have shown that the reaction is saturable with respect to both thrombin (KT = 3.6 x 10(-8) M) and antithrombin III (KAT = 1.0 x 10(-7) M) when the heparin concentration is low relative to the initial protein concentrations. The apparent first order rate constant for the rate-limiting step in the reaction was approximately 800 min-1. The reaction was subject to inhibition by antithrombin III/thrombin, the product of the reaction. Inhibition appeared to be noncompetitive with respect to antithrombin II with KP, the apparent heparin product dissociation constant, approximately equal to KT. When the heparin-enhanced antithrombin III/thrombin reaction was studied under conditions where the heparin concentration was high relative to the initial protein concentrations the overall reaction was second order. The initial reaction velocity, under any set of experimental conditions, could be described by the general rate equation for a random order bireactant, enzyme-catalyzed reaction, which is mathematically identical with the "template" model for the mechanism of action of heparin (Griffith, M. J. (1982) J. Biol. Chem. 257, 7360-7365).