The degradation of [125I]iodoinsulin (A14) by insulin protease (EC 3.4.22.11) was studied using HPLC. A reverse phase HPLC method is presented which allows the separation and quantitation of insulin degradation products. After incubation of [125I]iodoinsulin (A14) with insulin protease, there was an initial rapid loss of radioactivity from the [125I] iodoinsulin (A14) peak, which was quantitatively accounted for by the appearance of radioactivity in 11 different peaks, but was not accompanied by a proportional increase in the solubility of the sample in trichloroacetic acid. Two of the peaks showed appreciable accumulation before the others, and all but the first-eluted peak plateaued by 20 min. After 20 min of incubation, the amount of radioactivity present as the first-eluted peak, solubility in trichloroacetic acid, and insulin loss continued to increase at a steady, but slowed, rate. The order of appearance suggests that insulin protease acts on insulin in an ordered sequence of steps to generate a number of intermediates that are precipitable by trichloroacetic acid, but are subsequently degraded to material that is soluble in trichloroacetic acid. Sulfitolysis of 5 major peaks and subsequent HPLC analysis of the fragments showed none of the peaks to possess intact A chains. Peptide sequencing of 2 of the peaks indicates that the A-chain is cleaved in at least 2 positions, one beyond the 14th position, and one between the 13th and 14th amino acids (leucine and tyrosine).