The effects of cholinergic antagonists on vasopressin (VP) release were studied in an organ-cultured, compartmentalized, rat hypothalamo-neurohypophysial system which allows selective application of stimuli to either hypothala-mus or pituitary without disrupting axonal connections. Release of vasopressin from the neurohypophysis was measured by radioimmunoassay. Hexamethonium (10–5M) and atropine (5 × 10–5M) were tested both alone and in combination with hypothalamic osmotic stimulation (+15 mosm/kg H2O). In hypothalamus, neither hexamethonium nor atropine had any effect on basal VP release from pituitary. Hexamethonium, but not atropine, prevented the increase in VP release produced by increased osmolality of the hypothalamus side culture medium. In contrast, hexamethonium had no effect when applied to pituitary side, whereas atropine suppressed both basal and osmotically stimulated VP release. Atropine had no effect on basal or KC1-induced VP release in detatched neural lobes. Acetylcholine (Ach) (10–5M) to pituitary plus simultaneous hypothalamic stimulation (osmotic or 10–5M Ach) did not increase VP release above the hypothalamic stimulus alone. The results support a role for a hypothalamic excitatory nicotinic mechanism in osmoregulation. The presence of a muscarinic mechanism affecting VP release in pituitary was reconfirmed, but the data did not support the hypothesis that Ach stimulates VP release in pituitary by a presynaptic facilitatory m