Injection of 10 microgram of carbachol into a lateral cerebral ventricle caused a large reduction in urine flow and free water clearance and an increase in urine osmolality in water-loaded sheep. This antidiuretic effect persisted for at least 1 h, and was prevented by a prior intracerebroventricular (ICV) injection of atropine (100 microgram). ICV injections of atropine (100 microgram) given 10 min before and again midway throgh an intracarotid infusion of 2 M NaCl at 0.8 ml/min for 10 min did not alter the antidiuretic response which normally follows this hypertonic stimuli. It was concluded that in sheep, while periventricular cholinergic synapses capable of influencing antidiuretic hormone may exist, it is unlikely that ADH secretion in response to hypertonicity is mediated through such synapses. Cholinergic synapses located further from the cerebroventricular surface are not excluded from mediating the antidiuretic response to hypertonicity.