To determine whether oscillations in glycolysis could underlie the oscillations in O2 consumption observed in intact islets, we evaluated the capacity of an islet extract to exhibit spontaneous oscillations in glycolysis. When a cell-free extract obtained from ∼1,000 islets was supplied with glucose and glycolytic cofactors, oscillations in NADH fluorescence were obtained. After this demonstration of spontaneous oscillations in islet extracts, we bathed permeabilized clonal β-cells in the more plentiful spontaneously oscillating glycolytic muscle extract that generates pulses of α-glycerophosphate and pyruvate and induces oscillations in free Ca2+ and the ATP/ADP ratio. This preparation was used to investigate whether changes in Ca2+ and possibly α-glycerophosphate or pyruvate supply could underlie observed oscillations in O2 consumption and explain coordination between cytosolic and mitochondrial metabolism. We found that oscillations of O2 consumption and Ca2+ of a similar period were induced. Removal of medium Ca2+ with EGTA did not prevent the oscillations in O2 consumption nor were they greatly affected by the substantial rise in medium Ca2+ on treatment with thapsigargin to inhibit sequestration into the endoplasmic reticulum. The O2 oscillations were also not eliminated by the addition of relatively high concentrations of pyruvate or α-glycerophosphate. However, they were lost on addition of fructose-2,6-P2 at concentrations that prevent oscillations of glycolysis and the ATP/ADP ratio. Addition of a high concentration of ADP increased O2 consumption and also prevented O2 oscillations. These results suggest that the changes in respiration reflected in the O2 oscillations occur in response to the oscillations in the ATP/ADP ratio or ADP concentration and that this parameter is a primary regulator of O2 consumption in the pancreatic β-cell.