Uptake of 65Zn and distribution of 65Zn, total Zn, and insulin were measured in rat islets and islet granules under different conditions of islet culture. Specific activity of islet zinc (65Zn/Zn) was less than 15% that of extracellular Zn even after 48 h. Once in the islet, 65Zn approached 70% of equilibrium with granular Zn in 24 h and apparent equilibrium by 48 h. During a 24-h culture, at either high or low glucose, reduction of both islet Zn and insulin occurred. Zn depletion was greater than that predicted if Zn loss was proportional to insulin depletion and occurred only from the granular compartment, which represents only 1/3 of the total islet Zn. Extension of culture to 48 h caused additional insulin depletion, but islet zinc was unchanged. Omission of Ca during the 48-h culture caused a predicted increase in insulin retention, presumably by inhibiting secretion. Zn retention was not increased proportionately. Pretreatment of rats with tolbutamide caused a massive depletion of insulin stored in isolated islets, with little change in total islet Zn; subsequent culture of these islets resulted in a greater loss of granular Zn than predicted from the small loss of granular insulin. None of the conditions tested affected the percentage of either 65Zn or total Zn that was distributed in the islet granules. Zn exists in a metabolically labile islet compartment(s) as well as in secretory granules. Extragranular Zn, although not directly associated with insulin storage, may act as a reservoir for granular Zn and may regulate insulin synthesis, storage, and secretion in ways as yet unknown.