To evaluate the role of cholinergic neurones within the hypothalamus in modulating pancreatic-endocrine function, plasma levels of glucagon, insulin and glucose were measured after microinjection of acetylcholine into the ventromedial hypothalamic nucleus (VMH) of rabbits. Injection of acetylcholine (5 × 10–8 mol in 2 µl of saline) into the VMH of unanesthetized, unrestrained animals resulted in a prompt elevation of immunoreactive glucagon and glucose levels without any significant change in immunoreactive insulin level. The suppression of insulin secretion following cholinergic stimulation of the VMH was not counteracted by intravenous infusion of glucose (0.25 g/kg). Bilateral adrenalectomy prevented both hyperglucagonemia and hyperglycemia induced by the acetylcholine injection into the VMH. In adrenalectomized rabbits, exogenous glucose load was followed by a significant rise in the plasma insulin level despite the hypothalamic injection of acetylcholine. However, the response of insulin release to glucose load in these animals was much less than in adrenalectomized, saline-treated, control animals. These data support the conclusion that the action of acetylcholine within the hypothalamus on the pancreatic hormone secretions is mediated to a large part through sympatho-adrenomedullary activity. However, a part of the decreased insulin response to glucose may be mediated by direct innervation of the pancr