Challenge of the rat gastric mucosa with HCl stimulates intrinsic neurones in the myenteric plexus of the stomach as demonstrated by immunohistochemical detection of c-Fos. In multiple labelling experiments of whole-mounts and sections of the gastric corpus we determined the chemical code of the stimulated neurones and investigated further whether neural pathways involving capsaicin-sensitive afferents, cholinergic neurones or the vagal system contribute to the stimulation of these neurones. Intragastric (IG) administration of 0.5 m HCl caused c-Fos expression in 12% of myenteric neurones, whereas IG saline failed to induce c-Fos. All stimulated neurones stained for nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), but not for choline acetyltransferase (ChAT). Fibres coexpressing NOS/VIP/NPY were found predominantly in the external muscle layer and the muscularis mucosae of the stomach wall. Pretreatment with capsaicin or hexamethonium, combination of both pretreatments or vagotomy reduced HCl-induced c-Fos expression by 54%, 66%, 63% and 68%, respectively. The data indicate that mucosal acid challenge of the stomach stimulates inhibitory motor neurones in the myenteric plexus and that capsaicin-sensitive afferents as well as cholinergic neurones participate in the neuronal stimulation probably via a vago-vagal reflex.