The kidney plays a major role in the handling of circulating insulin in the blood, primarily via reuptake of filtered insulin at the luminal brush border membrane.125I-insulin associated with rat renal brush border membrane vesicles (BBV) in a time-and temperature-dependent manner accompanied by degradation of the hormone to trichloroacetic acid (TCA)-soluble fragments. Both association and degradation of125I-insulin were linearly proportional to membrane protein concentration with virtually all of the degradative activity being membrane assoicated. Insulin, proinsulin and desoctapeptide insulin all inhibited the association and degradation of125I-insulin by BBV, but these processes were not appreciably afected by the insulin-like growth factors IGF-I and IGF-II or by cytochrome c and lysozyme, low molecular weight, filterable, proteins, which are known to be reabsorbed in the renal tubules by luminal endocytosis. When the interaction of125I-insulin with BBV was studied at various medium osmolarities (300-1100 mos m) to alter intravesicular space, association of the ligand with the vesicles was unaffected, but degradation of the ligand by the vesicles decreased progressively with increasing medium osmolarity. Therefore, association of125I-insulin to BBV represented binding of the ligand to the membrane surface and not uptake of the hormone or its degradation products into the vesicles. Attempts to crosslink125I-insulin to a high-affinity insulin receptor using the bifunctional reagent disuccinimidyl suberate revealed only trace amounts of an125I-insulin-receptor complex in brush border membrane vesicles in contrast to intact renal tubules where this complex was readily observed. Both binding and degradation of125I-insulin by brush border membranes did not reach saturation even at concentrations of insulin approaching 10-5m. These results indicate the presence of low-affinity, high-capacity binding sites for125I-insulin on renal brush border membranes which can clearly distinguish insulin from the insulin-like growth factors and other low molecular weight proteins and polypeptides, but which do not differentiate insulin from its analogues ad do the biological receptors for the hormone. The properties and location of these binding sites make them attractive candidates for the sites at which insulin is reabsorbed in the renal tubule. © 1988 Springer-Verlag New York Inc.