Glucose stimulation of islets is coupled with the rapid intracellular release of myo-inositol 1,4,5-trisphosphate (IP3) and arachidonic acid which in turn mobilize Ca2+ stored in the endoplasmic reticulum (ER). The metabolism of glucose is required for insulin secretion although the link between glucose metabolism and the cellular events resulting in insulin release is unknown. In digitonin-permeabilized islets, glucose 6-phosphate (0.5-4 mM) increased significantly the ATP-dependent Ca2+ content of the ER at a free Ca2+ concentration of 1 microM. At 0.2 microM free Ca2+, glucose 6-phosphate (2-10 mM) had a smaller effect. Glucose, phosphate, mannose 6-phosphate, and fructose 1,6-diphosphate had no effect on the ATP-dependent Ca2+ content of the ER. Glucose 1-phosphate and fructose 6-phosphate also increased ATP-dependent Ca2+ content of the ER, presumably due to conversion to glucose 6-phosphate by islet phosphoglucomutase and phosphoglucoisomerase, respectively. The glucose 6-phosphate increase in the ATP-dependent Ca2+ content of the ER was shown to be mediated by glucose 6-phosphatase localized to the ER. Both arachidonic acid (10 microM) and the Ca2+ ionophore A23187 (2 microM) mobilized Ca2+ stored in the ER by glucose 6-phosphate. However, IP3-induced (10 microM) Ca2+ release from the ER was abolished in the presence of glucose 6-phosphate (0.5-10 mM). We propose that glucose 6-phosphate could provide a regulatory link between glucose metabolism and intracellular Ca2+ regulation by augmenting Ca2+ sequestered in the ER as well as attenuating IP3-induced Ca2+ release. Thus, glucose 6-phosphate would serve as an "off" signal leading to a decrease in intracellular Ca2+ when both the free Ca2+ and glucose 6-phosphate concentrations have increased following glucose stimulus.