Rat islets were maintained for 3 days in tissue culture at 2.8, 8.3, or 16.7 mM glucose, in the presence of [3H]leucine to label insulin stores. The immunoreactive insulin content of islets after 3 days at either 2.8 or 16.7 mM glucose was lower than that of islets exposed to 8.3 mM glucose. The specific radioactivity of stored labeled insulin, however, increased with increasing glucose. Islet insulin turnover was thus highest at 16.7 mM glucose. During a subsequent 1-day chase period at 2.8, 8.3, or 16.7 mM glucose for each of the three groups of islets prelabeled at these same three concentrations, all islets responded to glucose with increased insulin release. Release of immunoreactive insulin was highest at all three chase glucose concentrations for those islets preexposed for 3 days to 16.7 mM glucose. After 3-day labeling of islets at 8.3 mM glucose, there was an inverse relationship between the rate of release of labeled insulin and its intracellular degradation in response to the three glucose concentrations during the chase period. Those islets prelabeled at either 2.8 or 16.7 mM glucose showed much lower rates of intracellular degradation of labeled insulin stores during the chase period regardless of the ambient glucose or of the rate of release of the labeled hormone. Adaptive changes in islet insulin handling thus occurred during the 3-day maintenance period at different glucose concentrations. Whereas glucose concentration is, of course, thought to regulate insulin biosynthesis and release directly, regulation of degradation of stored insulin within B-cells appears to reflect both the relative activity of biosynthesis and release of the hormone and the insulin content of the islets. This last and striking relation is best interpreted as reflecting two labile, exchangeable, granule pools, one preferentially available for release and the other for intracellular degradation.