The binding of 125I-labeled insulin in thin sections of frozen fresh rat brain (95% specific binding) was shown using autoradiography. By several criteria including structure-activity relationship analysis, the brain insulin receptors were qualitatively indistinguishable from insulin receptors previously characterized on brain and other more typical target tissues and distinct from receptors for the insulin-like growth factors. The 125I-labeled insulin binding sites in brain formed a distinct pattern with high levels of binding in all olfactory areas and in closely related limbic regions. Binding was also prominent in the neocortex and the accessory motor areas of the basal ganglia and the cerebellum. Among ancillary brain structures only the choroid plexus demonstrated a high density of insulin receptors. When insulin receptors were located on cortical or laminated structures, the highest binding occurred in the superficial ("molecular" or "plexiform") layer, i.e. the layer which receives afferent input and/or is rich in the dendritic branches of principal neurons (e.g. external plexiform layer of the olfactory bulb and the molecular layer of the dentate gyrus). The enrichment of insulin receptors in the olfactory and limbic systems, which is a characteristic feature of other known neuropeptides, in addition to their prevalence in the strata occupied by the dendritic fields of principle neurons, suggests a neuromodulatory function for insulin in the brain.