Isolated rat pancreatic islets were perifused and analyzed for phosphate content immediately following the transient increase in the efflux of orthophosphate which occurs when insulin secretion is stimulated by glucose. In some instances, islets were perifused directly following isolation to minimize preparative delay; in others, islets were prelabeled during incubation with [32P]orthophosphate for 90 min prior to perifusion. In both experimental situations, total islet phosphate content declined 40--50% following exposure to stimulating concentrations of glucose and initiation of enhanced insulin release. In the experiments with prelabeled islets, tissue content of [32P]orthophosphate fell to a similar extent so that the specific radioactivity of islet orthophosphate was unaffected. Inhibited of heightened insulin release with Ni2+ did not modify the decrements in total or radioactive tissue orthophosphate, thus indicating that these responses to islet stimulation reflect events which are proximal to activated exocytosis. Simultaneous analyses for tissue ATP and ADP demonstrated that the efflux in orthophosphate and reduction in tissue orthophosphate content were not mediated via net changes in islet adenine nucleotides. The observations represent the first documentation that a net reduction of tissue inorganic phosphate is one of the early components of stimulus-secretion coupling in isolated pancreatic islets.