Normal glucose-stimulated insulin secretion is pulsatile, but the molecular mechanisms underlying this pulsatility are poorly understood. Oscillations in the intracellular free [ATP]/[ADP] ratio represent one possible mechanism because they would be expected to cause fluctuations in ATP-sensitive K(+) channel activity and hence oscillatory Ca(2+) influx. After imaging recombinant firefly luciferase, expressed via an adenoviral vector in single human or mouse islet beta-cells, we report here that cytosolic free ATP concentrations oscillate and that these oscillations are affected by glucose. In human beta-cells, oscillations were observed at both 3 and 15 mmol/l glucose, but the oscillations were of a longer wavelength at the higher glucose concentration (167 vs. 66 s). Mouse beta-cells displayed oscillations in both cytosolic free [Ca(2+)] and [ATP] only at elevated glucose concentrations, both with a period of 120 s. To explore the causal relationship between [Ca(2+)] and [ATP] oscillations, the regulation of each was further investigated in populations of MIN6 beta-cells. Incubation in Ca(2+)-free medium lowered cytosolic [Ca(2+)] but increased [ATP] in MIN6 cells at both 3 and 30 mmol/l glucose. Removal of external Ca(2+) increased [ATP], possibly by decreasing ATP consumption by endoplasmic reticulum Ca(2+)-ATPases. These results allow a model to be constructed of the beta-cell metabolic oscillator that drives nutrient-induced insulin secretion.