The rate and extent of insulin degradation by trypsin and alpha-chymotrypsin were examined in vitro, and the initial sites of cleavage by alpha-chymotrypsin were identified. The apparent Km for both enzymes was approximately the same but the apparent Vmax for alpha-chymotrypsin was 8.6 times greater. At a molar ratio of 172:1 (insulin:enzyme), chymotrypsin caused near-total loss of insulin within 40 min, while very little insulin was degraded by trypsin. Chymotrypsin appeared to cleave initially at the carboxyl side of the B26-Tyr and A19-Tyr residues, and additional cleavage at the B16-Tyr, B25-Phe, and A14-Tyr residue sites also occurred rapidly. Only two to three other susceptible bonds, which are not exposed at the surface of the insulin molecule, remained intact after the quenching of initial cleavage. Four of the amino acids involved in initial cleavage are essential for receptor binding ability, making it difficult to modify insulin chemically to achieve greater stability without losing activity.