beta-Cell-rich pancreatic islets from ob/ob mice were loaded with 45Ca in the presence of 20 mM glucose. When the islets were transferred to a nonradioactive incubation medium depleted of Ca2+, most of the 45Ca was lost during the first 10 min of incubation irrespective of whether or not glucose was present. Isolation of subcellular fractions under conditions minimizing 45Ca redistribution disclosed that the presence of 20 mM glucose during the nonradioactive incubation increased the retention of 45Ca in the mitochondrial fraction. This effect was not reproduced by K+ depolarization of the islets. Exposure of islets to 4 mM glucose failed to affect the total content of 45Ca in the islets but increased the incorporation of the isotope into cellular organelles. These results suggest that glucose, even at concentrations below the threshold for stimulation of insulin secretion, enhances the intracellular trapping of calcium.