In order to determine whether oxytocin release is controlled by an osmoreceptor mechanism identical with that for vasopressin release, the plasma oxytocin concentration and plasma osmolality were measured during intraatrial infusion and after intraventricular injection of various osmotic solutions in unanesthetized rats. Intraatrial infusion of 0.6 M NaCl Locke solution (L.S.) or 1.2 M mannitol L.S. elevated plasma oxytocin significantly, while 1.2 M urea L.S. caused only a small increase and isotonic L.S. did not change in plasma oxytocin. All hypertonic solutions produced significant and similar increases in the plasma osmolality. Plasma oxytocin was positively correlated with plasma osmolality in the animals infused with hypertonic NaCl or mannitol but not in the animals infused with hypertonic urea. The injection of 2 microliters of 0.6 M NaCl artificial cerebrospinal fluid (CSF) or 1.2 M mannitol CSF into the third ventricle caused a significant increase in plasma oxytocin immediately (5 min after injection) without changing plasma osmolality, while the intraventricular injection of 1.2 M urea CSF or isotonic CSF produced no significant change in plasma oxytocin. These results indicate that oxytocin release is controlled by osmoreceptors rather than Na receptors, that the adequate stimulus for the osmoreceptors is one which produces cellular dehydration and that the osmoreceptors are located in the brain region which is accessible to osmotic agents from both the outside and inside of the blood-brain barrier. Since the organum vasculosum of the lamina terminalis (OVLT) lacks a blood-brain barrier and is known to be involved in osmotic control of vasopressin release, a lesion was made in the anteroventral region of the third ventricle which encompasses the OVLT and the effect of hypertonic NaCl infusion on oxytocin release was examined. No significant increase in plasma oxytocin was observed after intraatrial infusion of 0.6 M NaCl L.S. in the lesioned rats. All of these findings lead to the conclusion that oxytocin release is under the control of osmoreceptors identical to those for vasopressin release.