The presence of specific, high affinity receptors for insulin has been demonstrated in purified preparations of bovine retinal microvessels. The binding of [125I]insulin to isolated retinal microvessels was inhibited by unlabeled insulin, but not by other peptide hormones. Scatchard analysis of the [125I]insulin binding data gave a curvilinear plot similar to that exhibited by insulin receptors in known insulin-sensitive tissues such as adipocytes and hepatocytes. Binding of [125I]insulin to retinal microvessels, followed by covalent cross-linking of the bound ligand to the alpha-subunit of the insulin receptor with the bifunctional reagent disuccinimidyl suberate, yielded a prominent specific [125I]insulin-labeled band when analyzed by sodium dodecyl sulfate-gel electrophoresis followed by autoradiography, and this band had a mobility identical to that of the corresponding complex obtained with rat liver plasma membranes (mol wt, 125,000). These results demonstrate for the first time that the retinal microvasculature, a major site of pathological injury in diabetes mellitus, contains insulin receptors that are similar to those present in known insulin-sensitive tissues, such as liver, fat, and muscle.