The molecular mechanism of the autolysis of rat α‐chymotrypsin B was investigated. In addition to the two already known autolytic sites, Tyr146 and Asn147, a new site formed by Phe114 was identified. The former two sites and the latter one are located in the autolysis and the interdomain loops, respectively. By eliminating these sites by site‐directed mutagenesis, their involvement in the autolysis and autolytic inactivation processes was studied. Mutants Phe114→Ile and Tyr146→His/Asn147→Ser, that had the same enzymatic activity and molecular stability as the wild‐type enzyme, displayed altered routes of autolytic degradation. The Phe114→Ile mutant also exhibited a significantly slower autolytic inactivation (its half‐life was 27‐fold longer in the absence and sixfold longer in the presence of Ca2+ ions) that obeyed a first order kinetics instead of the second order displayed by wild‐type chymotrypsin inactivation. The comparison of autolysis and autolytic inactivation data showed that: (a) the preferential cleavage of sites followed the order of Tyr146‐Asn147 → Phe114 → other sites; (b) the cleavage rates at sites Phe114 and Tyr146‐Asn147 were independent from each other; and (c) the hydrolysis of the Phe114‐Ser115 bond was the rate determining step in autolytic inactivation. Thus, it is the cleavage of the interdomain loop and not of the autolysis or other loops that determines the half‐life of chymotrypsin activity.