Thrombin (EC 220.127.116.11) has two exosites that mediate interactions between the enzyme and its substrates and cofactors. The binding of ligands to the exosites alters the functions of the protease, for example, when the cofactor thrombomodulin binds to both exosites I and II, it converts the enzyme from a procoagulant to an anticoagulant factor. It is unknown whether ligand binding to a thrombin exosite will alter the substrate specificity of the enzyme and thus contribute to the changed substrate repertoire of the enzyme upon engagement with cofactors. We first examined whether binding of ligands to exosites I and II altered the activity of the enzyme against fluorogenic peptide substrates. The efficiency of cleavage of substrates by thrombin did change when thrombomodulin or hirugen was present, indicating that exosite I occupancy changed the active site of the protease. The presence of heparin did not change the activity of the enzyme, indicating that exosite II occupancy had little effect on active site function. Investigation of the effects of exosite I occupancy by hirugen on thrombin specificity using phage display substrate libraries revealed that the ligand only changed the specificity of the enzyme to a small degree. Occupancy of both exosites by thrombomodulin induced greater changes to the specificity of the enzyme, with the prime side showing broader changes in amino acid frequencies. Thus, exosite I ligands do affect the activity and specificity of thrombin, but not greatly enough to explain the altered substrate profile of the enzyme when complexed with thrombomodulin.