We have monitored whole-cell and single channel ATP-sensitive K⁺ (K_ATP) currents in isolated rat glucagon-secreting pancreatic A-cells. Tolbutamide produced a concentration-dependent decrease in the whole-cell K_ATP conductance (K _i=6 µM) and initiated action potential firing. The K⁺ channel opener diazoxide, but not cromakalim or pinacidil, inhibited electrical activity and increased the whole-cell K⁺ conductance fourfold. ATP applied to the intracellular face of the membrane inhibited K_ATP channel activity with a K _i of 17 µM, an effect that could be counteracted by Mg-ADP and Mg-GDP. GTP and UTP did not affect K_ATP channel activity. Phosphatidylinositol 4,5-bisphosphate activated K_ATP channels inhibited by ATP after a delay of 90 s. In situ hybridisation demonstrated the expression of the mRNA encoding K_ATP channel subunits Kir6.2 and SUR1 but not Kir6.1 and SUR2. We conclude that rat pancreatic A-cells express K_ATP channels with the nucleotide-, sulphonylurea- and K⁺ channel-opener sensitivities expected for a channel formed by Kir6.2 and SUR1 subunits.