Epinephrine inhibits insulin release by activation of K(+) channels and subsequent hyperpolarization of pancreatic beta cells. The present study explored whether epinephrine-induced hyperpolarization is modified by phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositide-dependent kinase PDK1. Perforated patch-clamp was performed in islet cells isolated from PDK1 hypomorphic mice (pdk1(fl/fl)), expressing only 20% of PDK1, and in their wild-type littermates. At 16.8mM glucose, the cell membrane was hyperpolarized by epinephrine (1 microM), an effect significantly blunted in pdk1(fl/fl) and abrogated in wild-type cells by inhibition of PI3K with wortmannin (100 nM) or LY294002 (10 microM). The hyperpolarizing effect of epinephrine in pancreatic islet cells is thus sensitive to PI3K and PDK1.