The aim of the present study was to test the hypothesis that a creatine kinase/phosphocreatine system is involved in the regulation of K_ATP channels in pancreatic #-cells. The phosphocreatine concentration in isolated mouse islets clearly increased in parallel with the ATP/ADP ratio in response to a rise of the glucose concentration from 0.5 mM to 15 mM. The currents through K_ATP channels expressed in oocytes of Xenopus laevis were inhibited by injection of phosphocreatine or ATP but not by phosphate or creatine alone. In inside-out patches of #-cell membranes obtained from native #-cells, phosphocreatine reduced the open probability of single K_ATP channels in the presence of ADP but not in the absence of the nucleotide. These experiments suggest the existence of a K_ATP channel-associated creatine kinase that phosphorylates ADP. The creatine kinase inhibitor iodoacetamide suppressed the glucose-induced oscillations of the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_c. It is concluded that phosphocreatine serves as a shuttle for energy-rich phosphate from the mitochondria to the plasma membrane. The data provide a novel model for signal transduction to K_ATP channels in pancreatic #-cells.