Whether adenine nucleotides in pancreatic B cells serve as second messengers during glucose stimulation of insulin secretion remains disputed. Our hypothesis was that the actual changes in ATP and ADP are obscured by the large pool of adenine nucleotides (ATP/ADP ratio close to 1) in insulin granules. Therefore, mouse islets were degranulated acutely with a cocktail of glucose, KCl, forskolin, and phorbol ester or during overnight culture in RPMI-1640 medium containing 10 mM glucose. When these islets were then incubated in 0 glucose + azide (to minimize cytoplasmic and mitochondrial adenine nucleotides), their content in ATP + ADP + AMP was decreased in proportion to the decrease in insulin stores. After incubation in 10 mM glucose (no azide), the ATP/ADP ratio increased from 2.4 to > 8 in cultured islets, and only from 2 to < 4 in fresh islets. These differences were not explained by changes in glucose oxidation. The glucose dependency (0-30 mM) of the changes in insulin secretion and in the ATP/ADP ratio were then compared in the same islets. In nondegranulated, fresh islets, the ATP/ADP ratio increased between 0 and 10 mM glucose and then stabilized although insulin release kept increasing. In degranulated islets, the ATP/ADP ratio also increased between 0 and 10 mM glucose, but a further increase still occurred between 10 and 20 mM glucose, in parallel with the stimulation of insulin release. In conclusion, decreasing the granular pool of ATP and ADP unmasks large changes in the ATP/ADP ratio and a glucose dependency which persists within the range of stimulatory concentrations. The ATP/ADP ratio might thus serve as a coupling factor between glucose metabolism and insulin release.