Purified isolated rat renal glomeruli and tubules were incubated with [125I]insulin and the binding and degradation of the hormone were followed. Both glomeruli and tubules showed specific binding of [125I]insulin, which reached a plateau between 40 and 90 min of incubation. In the presence of increasing concentrations of unlabeled insulin ranging from 5-50,000 ng/ml, binding of [125I]insulin to the glomerular receptor(s) exhibited a higher affinity than that to the tubular receptor(s). Scatchad plots of the binding data were curvilinear, consistent with two or more classes of receptor sites in each preparation or negative cooperative site-site interactions. These plots supported a higher affinity for glomerular receptor(s) and a higher binding capacity for tubular receptor(s). Insulin analogues were less potent than insulin itself in inhibiting the binding of [125I]insulin to both glomeruli and tubules, and some were more effective in glomeruli than in tubules, again supporting the presence of receptor populations of differing affinity in the two preparations. Dissociation of bound [125I]insulin from the tubular fraction was more rapid than that from glomeruli also supporting a lower affinity of the receptors for insulin in this preparation.