Much of the afferent input thought to modulate vasopressin release from the magnocellular neuroendocrine cells of the supraoptic nucleus terminates in the region dorsal to the supraoptic nucleus. Cholinergic cells within this region may participate in the local processing of these afferent signals via synapses onto muscarinic cholinergic receptors. To investigate the role of these local synapses in vasopressin secretion, we characterized the muscarinic cholinergic influence on vasopressin secretion from the acute hypothalamoneurohypophysial explant in vitro. Acetylcholine induced a small dose-related secretion of vasopressin which could be totally blocked by atropine but not the nicotinic cholinergic antagonist, hexamethonium. Nicotine failed to release vasopressin from the explant, whereas alpha-bungarotoxin elicited a hypothalamic release of vasopressin which was atropine insensitive. Thus, local muscarinic receptors in the hypothalamus appear to participate in the control of neurohypophysial vasopressin secretion. The small magnitude of effect, however, is consistent with an indirect modulatory role rather than a major driving force for activation of the magnocellular neurons.