The present studies were designed to investigate the mechanism of previously-reported nocturnal hyperinsulinemia in the pinealectomized rat. Isolated islets were obtained from anesthetized control, sham-pinealectomized and pinealectomized rats, with 5 rats per surgical groups, during the early dark phase of the daily lightdark cycle. Batches of 3 islets each were incubated in various combinations of 2, 10 or 30 mM glucose with control buffer, medium in which cerebral cortex or pineal glands had previously been incubated for 2 hours, or sonicates of these same tissues. Insulin released into the culture medium was measured by radioimmunoassay. A significant hypersecretion of insulin was demonstrable in the islets from the pinealectomized animals. A stimulatory effect of both pineal medium and sonicates upon insulin release was similarly observed. Neither of these effects displayed an interaction with the concentration of glucose in the islet incubation medium and they, therefore, appear to be mediated by a mechanism which operates independently of stimulation by glucose. These results indicate that the rat pineal gland can exert direct effects upon insulin release from the islets, possibly through a humoral route. Further studies are in progress to characterize the nature and mode of action of the insulinotropic agent present in and released from the pineal gland.