Adenine nucleotides are thought to serve as second messengers in the control of beta-cell function by glucose, e.g. by regulating the activity of ATP-dependent K+ channels. However, their localization in different intracellular pools may mask the biologically relevant changes and complicate the interpretation of measurements in whole cells. The plasma membrane of mouse islet cells was selectively permeabilized by the alpha-toxin from Staphylococcus aureus to allow diffusion of cytoplasmic nucleotides. After permeabilization of cells from freshly isolated islets, approximately 68% of ATP, 45% of ADP, and 52% of AMP rapidly diffused out of the cells, whereas the insulin content hardly varied. The nondiffusible pool of nucleotides was stable for at least 90 min at 4 C, which suggests that it is contained in cellular organelles. The size of this nondiffusible pool decreased proportionally to insulin stores when these were lowered by stimulating secretion to different degrees during culture before permeabilization. From these results, it can be calculated that nondiffusible nucleotides are mainly contained in insulin secretory granules, with a small proportion in another, probably mitochondrial, compartment. Approximately 80% GTP and 30% GDP were present in the diffusible pool, and their relative proportions in the granular pool were only about 20% that of adenine nucleotides. Incubation of the cells in 20 instead of 2 mM glucose before permeabilization did not affect the nondiffusible pool, which indicates that the increase in the ATP/ADP ratio measured in intact cells occurred in the diffusible pool. Cytoplasmic nucleotide levels could be evaluated by subtracting the nondiffusible pool from the measurements in intact cells. It emerges that glucose induces large changes in the ATP/ADP ratio in the cytoplasmic pool, and that these changes are largely due to a fall in ADP.