The proteolytic degradation of porcine zinc insulin by α-chymotrypsin was previously found to depend markedly on the state of insulin aggregation (Pharm. Res. 9:864–869, 1992). In this study, the effect of bile salt-unsaturated fatty acid mixed micelles on α-chymotryptic degradation of insulin was further characterized. The incorporation of linoleic acid has greatly accelerated insulin degradation with the apparent first order rate constant being linearly related to the concentration of linoleic acid. At a 10 m M linoleic acid concentration solubilized in 10 m M sodium glycocholate, the proteoly tic degradation rate constant increased by 16 times, which could not be explained solely by the mechanism of insulin oligomer dissociation. Further, this effect is significantly reduced when the free carboxylic group of linoleic acid is methylated. The catalytic role of mixed micelles on chemical degradation of insulin was found to depend on the concentration of linoleic acid incorporated. When solubilized in the form of mixed micelles, linoleic acid chemically catalyzes peptide bond cleavage in a concentration-dependent manner.