Epinephrine inhibits insulin release by activation of K⁺ channels and subsequent hyperpolarisation of pancreatic beta cells. The present study explored the involvement of the PI3-kinse pathway. Pancreatic ß-cells were isolated from PDK1 hypomorphic mouse (pdk1^hm) or their wild type littermates (pdk1^wt). Ion channel activity was determined by whole cell recordings (pipette: 30 KCl, 95 K-gluconate, 1 MgCl₂, 3 EGTA, 1 CaCl₂; perfusate: 145 NaCl, 5 KCl, 2.5 CaCl₂, 1 MgCl₂, 0.1 tolbutamide, 0.1% BSA). Increase of perfusate glucose from 5 to 16.8 mM or K_ATP channel inhibition with tolbutamide (100mM) depolarized the cell membrane potential and triggered action potentials reflecting opening of Ca²⁺ channels. Under both, high glucose and tolbutamide, epinephrine (1 mM) within one minute hyperpolarised the cell membrane and decreased action potential frequency. The effect of epinephrine was abrogated by inhibition of phosphatidylinositol (PI)-3-kinase with LY294002 (10 mM) or with wortmannin (100 nM). Moreover, the hyperpolarization was significantly less pronounced in pdk1hm than in pdk1^wt mouse islets. In conclusion, epinephrine activates K⁺ channel activity in pancreatic ß-cells, an effect presumably contributing to its effect on insulin release.