Central administration of the preproglucagon-derived peptide glucagon-like peptide-1 significantly inhibits ingestion of food and water, and glucagon-like peptide-1 binding sites are present in a multitude of central areas involved in the regulation of ingestional behaviour. To evaluate further the neuroanatomical organization of central glucagon-like peptide-1 containing neuronal circuits with potential implications on ingestional behaviour, we carried out a series of experiments in the rat demonstrating the topographical sites of synthesis and processing of the preproglucagon precursor followed by a chromatographic analysis of the processed fragments. In situ hybridization histochemistry revealed that preproglucagon encoding messenger RNA was expressed in a single population of neurons in the caudal portion of the nucleus of the solitary tract. Gel chromatographic analysis of hypothalamic and brainstem tissue extracts revealed that the preproglucagon precursor is processed in a fashion similar to that seen in the small intestine, preferentially giving rise to glicentin, glucagon-like peptide-1 and glucagon-like peptide-2. This single brain site of glucagon-like peptide-1 synthesis was subsequently confirmed by immunohistochemical demonstration of glucagon-like peptide-1-immunoreactive perikarya in the central and caudal parts of the nucleus of the solitary tract. Numerous sites containing glucagon-like peptide-1 immunoreactive fibres were, however, discovered in the forebrain including hypothalamic, thalamic and cortical areas. The densest innervation by glucagon-like peptide-1 immunoreactive nerve fibres was seen in the hypothalamic dorsomedial and paraventricular nuclei, but numerous glucagon-like peptide-1 immunoreactive fibres were also seen throughout the periventricular strata of the third ventricle. Dual-labelling immunohistochemistry for tyrosine hydroxylase and glucagon-like peptide-1 gave no evidence for co-localization of catecholamines and glucagon-like peptide-1 in neurons of the lower brainstem. To identify neurons of the nucleus of the solitary tract that project to the hypothalamic paraventricular nucleus, the retrograde tracer FluoroGold was injected into this hypothalamic target and dual immunocytochemical identification of glucagon-like peptide-1 and tyrosine hydroxylase-positive neurons was performed on brainstem sections containing retrogradely labelled perikarya. From this experiment it was seen that many of the retrogradely labelled neurons in the central portion of the nucleus of the solitary tract are catecholaminergic, while none is glucagon-like peptide-1 immunoreactive. In contrast, most of the retrogradely labelled neurons of the caudal portion of the nucleus of the solitary tract contain glucagon-like peptide-1. These observations further substantiate that glucagon-like peptide-1 neurons of the solitary tract constitute a distinct non-catecholaminergic cell group which projects to many targets, one of which is the hypothalamic paraventricular nucleus.