FMRFamide stimulated, in a dose-dependent manner, the efflux of [14C]acetylcholine (ACh) from isolated synaptosomes of guinea-pig ileum preloaded with labelled choline. Participation of the cholinergic mechanism and/or substance P was ruled out by the finding that antagonists failed to affect the FMRFamide-induced release of ACh. The ACh-releasing action of FMRFamide was negated by the deletion of calcium ion from the bathing medium and it was also abolished by tetrodotoxin. The results obtained suggest that FMRFamide possesses the ability to induce the release of ACh from enteric synaptosomes of guinea-pig.