We previously isolated a monoclonal antithrombin IgG from a patient with multiple myeloma [Colwell et al. (1997) Br. J. Haematol. 97, 219-226]. Using a panel of 55 surface mutants of recombinant thrombin, we now show that the epitope for the IgG most likely includes Arg-101, Arg-233, and Lys-236 in exosite II. The IgG affects the rate at which thrombin cleaves various peptide p-nitroanilide substrates with arginine in the P1 position, increasing the kcat for substrates with a P2 glycine residue but generally decreasing the kcat for substrates with a P2 proline. The allosteric effect of the IgG is altered by deletion of Pro-60b, Pro-60c, and Trp-60d from the 60-loop of thrombin, which lies between exosite II and the catalytic triad. The effect of the IgG, however, does not depend on the presence or absence of sodium ions, a known allosteric regulator of thrombin. The IgG does not affect the conformation of thrombin exosite I as determined by binding of a fluorescent derivative of hirudin54-65. These results provide evidence for a direct allosteric linkage between exosite II and the catalytic site of thrombin.