To characterize the neural pathways involved in lower esophageal sphincter relaxation, intraluminal pressures from the lower esophageal sphincter of the opossum were monitored during swallowing, vagal efferent nerve stimulation, and intraluminal balloon distention in the presence and absence of pharmacologic antagonism of putative neurotransmitters. The combination of atropine, hexamethonium, and 5-methoxydimethyltryptamine, which is known to block ganglionic transmission in the vagal inhibitory pathway to the lower esophageal sphincter, significantly antagonized LES relaxation induced by both swallowing and vagal stimulation, but did not affect the LES relaxation induced by balloon distention. Administration of the nitric oxide synthase inhibitor Nωnitro-L-arginine methyl ester, on the other hand, markedly inhibited LES relaxation induced by vagal stimulation, swallowing, and balloon distention, and this effect was reversed by administration of the nitric oxide synthase substrate L-arginine. These studies indicate that the distension-induced intramural pathway mediating LES relaxation does not involve ganglionic transmission similar to that of the vagal inhibitory pathway to the LES. However, the LES relaxation induced by all forms of stimuli appears to depend on nitric oxide as a final mediator.Key words: ganglionic transmission, muscarinic, nicotinic, serotonergic, nitric oxide.