Orexins and orexin-receptors are localized by displaying their immunoreactivity in the enteric nervous system. Intracellular recordings were made from isolated myenteric neurons to investigate actions of orexin-A in the myenteric plexus of the guinea-pig ileum. Superfusion of orexin-A caused membrane depolarizations in a subset of S and AH neurons. Orexin-A responses were preserved in Ca2+ free/high Mg2+ solution and associated with an increase in input membrane resistance; their reversal potential was about -90 mV. Orexin-A augmented nicotinic fast EPSPs, whereas it did not affect the postsynaptic sensitivity to acetylcholine; this indicates that orexin-A increased the presynaptic release of acetylcholine. In conclusion, orexin-A contributes in the regulation of gut motility via its pre- and postsynaptic actions in the myenteric plexus.