In the present study we aimed to discretely characterise ganglionic and neuroeffector transmission to the ferret lower oesophageal sphincter (LOS) using a novel preparation of LOS muscle with intact vagal innervation in conjunction with isolated LOS muscle strips. In this way we could compare vagally mediated LOS relaxation with that of enteric inhibitory motorneurones which were directly stimulated. Preparations of LOS muscle, with or without attached vagus nerves, were dissected from adult ferrets and maintained under preload in organ baths, where LOS muscle developed spontaneous tone. LOS relaxations in response to vagal stimulation (0.5-5 Hz, 30 V) were recorded, alone and following pretreatment with tetrodotoxin (TTX), hexamethonium (Hex), Hex and atropine and NG-nitro-L-arginine (L-NNA). Direct activation of enteric inhibitory motorneurones was performed via electrical field stimulation (EFS). Vagal stimulation elicited frequency-dependent relaxations of the LOS that were abolished by tetrodotoxin (1 microM) and markedly reduced following L-NNA pretreatment (100 microM), but unaltered following pretreatment with the selective VIP or PACAP receptor antagonists VIP (10-28) or PACAP (6-38), respectively (each at 5 microM). The potent NOS inhibitor S-methyl-L-thiocitrulline (100 microM) inhibited LOS relaxation to the same degree at 5 Hz. Hex alone (500 microM) reduced maximal relaxation by 50%; in combination with atropine (2 microM), relaxation was almost abolished. In isolated LOS muscle strips, neither VIP (10-28) nor PACAP (6-38) altered EFS-induced relaxation. Taken together, these results suggest ganglionic neurotransmission to the ferret LOS occurs mainly through a combination of nicotinic and muscarinic receptors and utilises nitroxidergic enteric inhibitory motorneurones to relax the LOS. Moreover, LOS relaxation due to direct activation of inhibitory motorneurones also utilises primarily nitric oxide and other as yet undefined neurotransmitters. Neither VIP nor PACAP are involved in vagally mediated or direct enteric neuronally stimulated LOS relaxation in the ferret.