ANTITHROMBIN III (heparin cofactor) is known to inhibit thrombin¹ and Factor X_a^1–3 (activated Factor X). We have purified antithrombin⁴ from human plasma by a series of chromatographic and electrophoretic separation techniques. The homogeneity of the final product is demonstrated by disc gel electrophoresis, SDS gel electrophoresis and immunoelectrophoresis⁴. Using this preparation, we have shown that antithrombin forms a 1 : 1 stoichiometric complex with thrombin which cannot be dissociated with denaturing and reducing agents. Addition of heparin dramatically accelerates the rate of formation of this complex. Complex formation is completely dependent on an interaction between the serine active centre of thrombin and an arginine reactive site of antithrombin. Furthermore, ε-amino lysyl groups of antithrombin serve as binding sites for the highly negatively charged heparin. Based on these data and other evidence, we have proposed that heparin acts to accelerate inhibitor function by binding to antithrombin and inducing an allosteric modification in it, which renders the arginine in its reactive site more accessible to the serine in the active centre of thrombin⁴.