Less than a stoichiometric amount of bovine chymotrypsin was progressively inactivated by a bovine AT III preparation. The rate of inactivation was not influenced by a synthetic substrate. Since chymotrypsin has low affinity for heparin, the polysaccharide only doubled the inactivation of the enzyme by the AT III preparation. At a low salt concentration, about two-thirds of the chymotrypsin was instantaneously inactivated by the AT III preparation in the presence of heparin. These observations indicate that inactivation of chymotrypsin is due to AT III itself, not to another contaminating inhibitor(s).