A portion of medial basal hypothalamus containing the supraoptic nuclei with the neurohypophysis attached was organ cultured. Hypothalamus and neurohypophysis were maintained in separate compartments, and the intact infundibular stalk passed through a hole in a fluid-tight barrier which separated the two compartments. After 24, 48 and 72 h in culture, vasopressin (VP) release from the neurohypophysis was measured during a control hour and again during an immediately subsequent test hour. Test hour VP release was expressed as a percentage of control hour release. Test substances were added to either the pituitary or the hypothalamus compartment. Acetylcholine stimulated pituitary VP release both when added to hypothalamus (10–5M) and when added directly to neural lobe (10–6 M and above). Acetylcholine 10–5 M and no effect when isolated neural lobes (severed from hypothalamus to culture) were similarly tested. Hexamethonium blocked the stimulation of pituitary VP release evoked by addition of acetylcholine to hypothalamus. However, in pituitary, atropine prevented the stimulatory effect of acetylcholine. Atropine had no effect on VP release from severed neural lobes. These data show that high concentrations of acetylcholine can stimulate VP release from pituitary both by a hypothalamic action and also by a direct effect in neural lobe. Further, a nicotinic cholinergic receptor mediates the action of acetylcholine in hypothalamus whereas a muscarinic cholinergic receptor is involved in the direct pituitary response to acetylcholine. Intact axonal connections between hypothalamus and pituitary are required in order for acetylcholine to stimulate VP release in neurohypop