Insulin-binding sites were localized in the rat brain by means of light microscopic radioautography. Male rats were injected intracardially with [¹²⁵I]iodoinsulin in the absence and presence of excess unlabeled insulin, insulin analogs, or structurally unrelated polypeptide hormones. Five minutes after injection, the brains were preserved in vivo by perfusion and subjected to radioautographic procedures. Radioautographic reactions caused by [¹²⁵I]iodoinsulin occurred over all circumventricular organs lacking a blood-brain barrier. In addition, the medial basal hypothalamus, the medial paravagal region, and the choroid plexus were labeled. In the presence of a 500-fold excess of coinjected unlabeled insulin, the radioautographic reactions over the circumventricular organs, the medial basal hypothalamus, and the paravagal region were reduced by 56–78%, indicating the presence of competitive binding sites. In the external contact zone of the median eminence and in the hypothalamic arcuate nucleus, the effect of increasing concentrations of unlabeled insulin, analogs, and structurally unrelated peptides on the radioautographic reaction was studied in detail. Increasing amounts of unlabeled insulin caused a progressive decrease in radioautographic reaction. The competitive effect of insulin analogs was parallel to their known biological potency, and structurally unrelated substances were noninhibitory. This study demonstrates the presence of binding sites for vascular insulin in the circumventricular organs, regions associated with glucosemonitoring function, and in the medial basal hypothalamus and paravagal region, areas implicated in satiety and gastrointestinal regulation. The preferential localization of insulin-specific binding sites to the external region of the median eminence raises the possibility that insulin acts on tuberoinfundibular nerve terminals to influence directly the release of hypophysiotropic substances.