1. The rate of release of neurohypophysial hormones in vitro, using isolated, halved neural lobes of the rat in an incubation medium containing excess K(+) and Ca(2+), was measured. The highest average rate of release was observed between 10 and 20 min after commencement of incubation.2. Incubation of isolated, halved rat neural lobes in the presence of acetylcholine, with or without eserine, did not stimulate hormone release. When complete isolated hypothalamo-neurohypophysial systems were incubated in a suspension medium containing 10(-7) mg/ml. acetylcholine a significant increase in the release of oxytocin occurred (P < 0.01); the increase in vasopressin release was less pronounced (P < 0.05).3. Uptake of O(2) by the isolated, halved neural lobes and the hypothalamo-neurohypophysial systems continued for 2-3 hr, i.e. in excess of the experimental incubation time.4. During the first 40 min of incubation the control halved neural lobes increased in weight; the neural lobes incubated in buffer containing high potassium and calcium showed no increase in weight.5. Neural lobes incubated in buffer containing excess K(+) and Ca(2+) contained about 3 times as much potassium as controls. The sodium content was not affected significantly.6. Factors involved in the process of neurohypophysial hormone release are discussed.