Several in vitro models of gastric relaxation have elucidated a role of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) in non-adrenergic, non-cholinergic (NANC) vagally mediated gastric relaxation. However, these models do not necessarily mimic the events leading to gastric relaxation in the whole animal. We have recently described a vagally mediated gastric relaxation evoked by micro-injection of substance P (SP) into the nucleus raphe obscurus (NRO). The present study was performed to elucidate whether this CNS-stimulated in vivo gastric relaxation involved acetylcholine, NO and VIP. Atropine (1 mg kg-1 i.v.), reduces both the rapid nadir and sustained gastric relaxation evoked by SP in the NRO, and the residual responses are abolished by NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 10 mg kg-1 i.v.), an NO synthase inhibitor. Blockade of NO synthase alone is not sufficient to abolish the effect of SP into the NRO on intragastric pressure. A VIP antagonist, [p-chloro-D-Phe6, Leu17]VIP (32 micrograms i.v.) alone, or with the addition of L-NAME, does not affect the nadir of the gastric relaxation in response to SP microinjected into the NRO; however, both antagonists reduce the CNS-evoked sustained intragastric pressure relaxation. We conclude that, in CNS-evoked gastric relaxation, inhibition of cholinergic pathways is potentially important for both the rapid nadir and sustained gastric relaxation, and both NO and VIP contribute to sustained gastric relaxation.