Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent proglucagon-derived hormone that stimulates intestinal growth through poorly understood paracrine and/or neural pathways. The relationship between GLP-2 action and a vagal pathway is unclear. Our aims were to determine whether 1) the GLP-2 receptor (GLP-2R) is expressed on vagal afferents by localizing it to the nodose ganglia; 2) exogenous GLP-2 stimulates the vagal afferent pathway by determining immunoreactivity for c-fos protein in the nucleus of the solitary tract (NTS); and 3) functional ablation of vagal afferents attenuates GLP-2-mediated intestinal growth in rats maintained with total parenteral nutrition (TPN). A polyclonal antibody against the N terminus of the rat GLP-2R was raised and characterized. The GLP-2R was localized to vagal afferents in the nodose ganglia and confirmed in enteroendocrine cells, enteric neurons, and nerve fibers in the myenteric plexus using immunohistochemistry. Activation of the vagal afferent pathway, as indicated by c-fos protein immunoreactivity in the NTS, was determined by immunohistochemistry after ip injection of 200 microg human GLP-2. GLP-2 induced a significant 5-fold increase in the number of c-fos protein immunoreactive neurons in the NTS compared with saline. Ablation of vagal afferent function by perivagal application of capsaicin, a specific afferent neurotoxin, abolished c-fos protein immunoreactivity, suggesting that activation of the NTS due to GLP-2 is dependent on vagal afferents. Exogenous GLP-2 prevented TPN-induced mucosal atrophy, but ablation of vagal afferent function with capsaicin did not attenuate this effect. This suggests that vagal-independent pathways are responsible for GLP-2 action in the absence of luminal nutrients during TPN, possibly involving enteric neurons or endocrine cells. This study shows for the first time that the GLP-2R is expressed by vagal afferents, and ip GLP-2 activates the vagal afferent pathway.