Closure of ATP-sensitive K+ (K(ATP)) channels is part of the stimulus-secretion coupling mechanism in the pancreatic beta-cell, leading to membrane depolarization and influx of Ca2+ through voltage-sensitive L-type Ca2+ channels. The elevated ATP/ADP ratio seen in the presence of high levels of glucose has been postulated to mediate the glucose-induced closure of the K(ATP) channels and rise in cytoplasmic free Ca2+ concentration ([Ca2+]i), or alternatively to be a consequence of activation of mitochondrial dehydrogenases by the increase in [Ca2+]i. To distinguish between these two possibilities, the time course of the change in the ATP/ADP ratio was determined in comparison with that of [Ca2+]i. We here show that a severalfold rise in the ATP/ADP ratio occurs rapidly on stimulation of suspensions of mouse pancreatic beta-cells with glucose. The change in the ATP/ADP ratio is an early event that begins within 20-40 s and precedes the rise in [Ca2+]i. The temporal relationship indicates that the adenine nucleotide changes cannot be a consequence of the [Ca2+]i changes and may indeed be the connecting link between glucose metabolism and [Ca2+]i changes. When the cells were sequentially treated with high glucose concentration, clonidine and finally high extracellular Ca2+ concentration to induce synchronized oscillations in [Ca2+]i in the cell suspension, corresponding oscillations in the ATP/ADP ratio were observed. Glucose 6-phosphate levels oscillated out of phase with the ATP/ADP ratio. These results support the hypothesis that the Ca2+ oscillations previously observed in glucose-stimulated single islets or beta-cells may reflect oscillations in the ATP/ADP ratio that accompany oscillatory glycolysis.