Intracerebroventricular (i.c.v.) injection of choline (50-150 μg), a precursor of the neurotransmitter acetylcholine, produced a time-and dose-dependent increase in plasma vasopressin levels in conscious, freely moving rats. The increase in plasma vasopressin in response to i.c.v. choline (150 μg) was inhibited by pretreatment with the nicotinic receptor antagonist, mecamylamine (50 μg; i.c.v.), but not by the muscarinic receptor antagonist, atropine (10 μg; i.c.v). The choline-induced rise in plasma vasopressin levels was greatly attenuated by hemicholinium-3 (HC-3; 20 μg; i.c.v.), a neuronal choline uptake inhibitor. Choline (50 or 150 μg; i.c.v.) produced a much greater increase in plasma vasopressin levels in osmotically stimulated or hemorrhaged rats than in normal rats. Choline (150 μg; i.c.v.) also enhanced plasma vasopressin response to graded hemorrhage; the enhancing effect of choline was also attenuated by HC-3 (20 μg; i.c.v.). Choline and acetylcholine concentrations in hypothalamic dialysates increased significantly following i.c.v. injection of choline (150 μg). It is concluded that choline increases plasma vasopressin levels by stimulating central nicotinic receptors indirectly, through the enhancement of acetylcholine synthesis and release, and augments the ability of osmotic stimulations or hemorrhage to stimulate vasopressin release.