The therapeutically important anticoagulant heparin catalyzes inactivation of thrombin by antithrombin III via formation of an intermediary ternary thrombin-heparin-antithrombin III complex that is subsequently converted to a stable thrombin-antithrombin III complex with the release of heparin. Point mutations at Arg-180, Arg-245, Lys-248, and Lys-252 in thrombin markedly reduced the efficiency of heparin catalysis by decreasing the stability of the ternary intermediate, whereas the inactivation of thrombin by antithrombin alone was not affected by these mutations. These results together with an analysis of the x-ray crystal structure of thrombin yielded a model for the thrombin-heparin interaction, wherein heparin forms salt linkages along a groove in thrombin defined by Lys-252, Lys-248, Arg-245, Arg-89, Arg-98, and Arg-180.