To examine the possibility that ATP modulates insulin secretion by an autocrine mechanism, we measured the local concentration of released ATP at the surface of a single pancreatic β cell by a new biosensor technique, using PC12 cells expressing ligand-gated cation channels, P2X₂ receptors. Upon application of glucose or glibenclamide, a series of current spikes, whose amplitude equates to an ATP concentration of over 25 µM, were recorded from a PC12 cell using the whole-cell patch-clamp technique, when placed near a rat pancreatic β cell at 37°C. The current response was inhibited by cooling (below 30°C) or by applying an ATP-hydrolysing enzyme (apyrase) or a P2 receptor blocker (suramin). Thus, it is concluded that pancreatic β cells secrete ATP in response to glucose stimulation, thereby increasing the ATP concentration close to the cell surface sufficiently high enough to enhance insulin secretion from the pancreatic β cells.