The degradation of insulin in isolated liver endosomes and the relationships of this process with ATP-dependent endosomal acidification have been studied. Incubation of endosomal fractions containing 1251-insulin in isotonic KCl at 30°C resulted in a rapid loss of insulin integrity as judged from trichloroacetic acid precipitability, Sephadex G-50 chromatography, immunoreactivity and receptor binding ability, with a maximum at pH 5 -6 ( t 1 , 2 : 10, 10, 6 and 6 min, respectively). On a log/log plot, the amount of acid-soluble products generated was linearly related to the amount of insulin associated with endosomes (slope, 0.80). Upon incubation, virtually all acid-soluble products diffused out of endosomes as judged from their solubility in aqueous poly(ethyleneglyco1). In permeabilized endosomes, intact insulin was also released in part extraluminally, but only when degradation was inhibited did this release increase with lowering pH. ATP shifted the pH for maximal insulin degradation to about 7.5 -8.5 and caused endosomal acidification as judged from the uptake of acridine orange and the fluorescence of internalized fluorescein-labeled dextran and galactosylated bovine serum albumin (dpH about 0.8 -0.9). GTP, ITP and UTP exerted comparable effects but with lower potencies. The ability of ATP to alter the pH dependence of insulin degradation was maximal in the presence of CI-, other anions being less effective (Br-> gluconate = SO:-> NO; = sucrose = mannitol) and/or inhibitory (NO;). Na', K + and Li' supported more effectively ATP-dependent insulin degradation than did choline. Divalent cations were required for the ATP effect (Mg2Little or no effects of ATP occurred in the presence of proton ionophores such as monensin and carbonyl cyanide chlorophenylhydrazone, and inhibitors of the proton ATPase such as N-ethylmaleimide. The abilities of nucleotides, ions and inhibitors to support or inhibit ATP-dependent insulin degradation were well correlated with their abilities to affect ATP-dependent acidification. The acidotropic agents chloroquine and quinacrine caused a leftward shift in the pH dependence of insulin degradation and a decrease in maximal degradation; in the presence of ATP, chloroquine almost completely inhibited degradation at pH 5 -9. It is concluded that ATP-dependent acidification, in part by enhancing the dissociation of the insulinreceptor complex, is required for optimum degradation of insulin within liver endosomes.Biochemical and morphological studies on intact liver and isolated hepatocytes have shown that, following binding to its receptor at the cell surface, insulin undergoes rapid endocytosis along with its receptor (reviewed in [l -31). Subsequently, the receptor is recycled in part to the cell surface, while most of the insulin is degraded within the cell. Several lines of evidence indicate that, although initially thought to occur in lysosomes, degradation of internalized insulin occurs mainly in endosomes. First, little association of insulin with authentical lysosomes is demonstr...