Isolated pancreatic islets were used to determine whether substances of hypothalamic origin could directly influence the release of insulin and glucagon. Media in which various regions of the brain had been incubated were tested in the islet system, as were the synthetic peptides neurotensin and substance P, and the catecholamines, dopamine and norepinephrine. Substance(s) released from the ventromedial hypothalamic (VMH) segments in vitro inhibited insulin release and stimulated glucagon release from the islets. Incubates of ventrolateral hypothalamic (VLH) or cortex tissue failed to alter insulin or glucagon levels. The VMH medium retained these activities even after oxidation with K3Fe (CN)6, whereas the ability of the catecholamines to inhibit insulin release and stimulate glucagon release was eliminated by this treatment. Neurotensin and substance P (0.1 and 1.0 nmol/ml) inhibited insulin release while glucagon release was increased; however, radioimmunoassay indicated that these peptides were virtually absent from the VMH incubate. These results show that incubates of VMH contain substances which can inhibit insulin and stimulate glucagon release in vitro. They may influence the endocrine pancreas by way of the peripheral circulation although the possibility of their occurrence in or near the pancreas itself has not been excluded.