The substrate specificity of thrombin is regulated by binding of macromolecular substrates and effectors to exosites I and II. Exosites I and II have been reported to be extremely linked allosterically, such that binding of a ligand to one exosite results in near-total loss of affinity for ligands at the alternative exosite, whereas other studies support the independence of the interactions. An array of fluorescent thrombin derivatives and fluorescein-labeled hirudin54-65 ([5F] Hir54-65(SO3-)) were used as probes in quantitative equilibrium binding studies to resolve whether the affinities of the exosite I-specific ligands, Hir54-65(SO3-) and fibrinogen, and of the exosite II-specific ligands, prothrombin fragment 2 and a monoclonal antibody, were affected by alternate exosite occupation. Hir54-65(SO3-) and fibrinogen bound to exosite I with dissociation constants of 16-28 nM and 5-7 μM, respectively, which were changed ≤2-fold by fragment 2 binding. Native thrombin and four thrombin derivatives labeled with different probes bound fragment 2 and the antibody with dissociation constants of 3-12 μM and 1.8 nM, respectively, unaffected by Hir54-65(SO3-). The results support a ternary complex binding model in which exosites I and II can be occupied simultaneously. The thrombin catalytic site senses individual and simultaneous binding of exosite I and II ligands differently, resulting in unique active site environments for each thrombin complex. The results indicate significant, ligand-specific allosteric coupling between thrombin exosites I and II and catalytic site perturbations but insignificant inter-exosite thermodynamic linkage.