1. Intraluminal pressure was recorded from the isolated guinea-pig and mouse stomach with the vagus and sympathetic nerves attached.2. The response to vagal stimulation, which consists of an excitatory and an inhibitory component, resembled the response to 5-hydroxytryptamine (5-HT), which has no direct action on the muscle but acts on intrinsic excitatory and inhibitory ganglia.3. In the presence of hyoscine, the effect of vagal stimulation, of nicotinic compounds and of 5-HT were all purely relaxant. Competitive block of ganglionic receptors for acetylcholine reduced the vagal relaxation without antagonizing 5-HT. Specific desensitization of ganglionic receptors for 5-HT reduced the vagal relaxation without antagonizing nicotinic compounds.4. During the early phase of the blocking action of nicotine, responses to vagal stimulation and to 5-HT were both abolished. As the non-specific antagonism changed to the later phase of specific antagonism to acetylcholine, the inhibitory (but not the excitatory) component of the vagal response recovered partially, in parallel with the recovery of the relaxant effect of 5-HT.5. The vagal inhibitory effect was completely abolished only when competitive block of acetylcholine receptors was combined with desensitization of 5-HT receptors.6. Stimulation of the mouse stomach (after asphyxiation of the mucosa and exclusion of the luminal content) in the presence of hyoscine caused the release of 5-HT; this release was blocked by tetrodotoxin.7. The results, together with previous observations that 5-HT is contained within preganglionic nerve fibres in the myenteric plexus, are consistent with the hypothesis that 5-HT, with acetylcholine, may be a neurotransmitter in the vagal inhibitory innervation of the stomach.