Glucagon-like peptide-1 (GLP-1) immunoreactivity has been found in autonomic and neuroendocrine brain regions, whereas only limited data are available regarding the characterization and localization of brain GLP-1 receptors. In the present study, using quantitative in vitro autoradiography, a high density of specific binding sites for GLP-1 was characterized on sections of the posterior pituitary lobe of the rat. Low specific binding of radiolabeled GLP-1 was found in the anterior lobe and no specific binding in the intermediate lobe. To examine the specificity of GLP-1 binding sites, sections of the posterior lobe were incubated with radiolabeled GLP-1 in the presence of various peptides. Radiolabeled [Tyr³⁹]exendin-4, a specific GLP-1 agonist, bound to these receptor sites with the same affinity as GLP-1, while glucagon and vasoactive intestinal peptide (VIP) were unable to displace ¹²⁵I-GLP-1. Both unlabeled exendin-4 and GLP-1 inhibited this binding with equally high affinity. Using ¹²⁵I-[Tyr³⁹]exendin-4 as radiolabel, the concentration of binding sites was found to be 7.8 ± 0.4 fmol/mg tissue. Further analysis of the binding data from experiments with tissue slices revealed the presence of high and low affinity binding sites. In experiments with unlabeled [Tyr³⁹]exendin-4, the K_ds were 6.2 ± 1.4 × 10^–12 and 9.3 ± 1.5 × 10^–10M, respectively, and in experiments with unlabeled GLP-1, 3.4 ± 1.8 × 10-¹² and 5.9 ± 1.5 × 10^-10M, respectively. Since both GLP-1 and [Tyr³⁹]exendin-4 reveal the same binding characteristics, it is concluded that both peptides bind to the same receptor sites in the rat posterior pituitary lobe. ¹²⁵I-GLP-1 and ¹²⁵I-[Tyr³⁹]exendin-4 also specifically bound to the nucleus of the solitary tract, the subfornical organ, the median eminence, the lateral mammillary nucleus and the lateral subnucleus of the interpeduncular nucleus. The presence of GLP-1-binding sites in different circumventricular organs suggests that this hormone may serve to modulate autonomic and neuroendocrine functions in the rat brain.