This study examines the effect of epinephrine, a known physiological inhibitor of insulin secretion, on the membrane potential of pancreatic islet cells from sulfonylurea receptor-1 (ABCC8)-null mice (Sur1KO), which lack functional ATP-sensitive K+(KATP) channels. These channels have been argued to be activated by catecholamines, but epinephrine effectively inhibits insulin secretion in both Sur1KO and wild-type islets and in mice. Isolated Sur1KO β-cells are depolarized in both low (2.8 mmol/l) and high (16.7 mmol/l) glucose and exhibit Ca2+-dependent action potentials. Epinephrine hyperpolarizes Sur1KO β-cells, inhibiting their spontaneous action potentials. This effect, observed in standard whole cell patches, is abolished by pertussis toxin and blocked by BaCl2. The epinephrine effect is mimicked by clonidine, a selective α2-adrenoceptor agonist and inhibited by α-yohimbine, an α2-antagonist. A selection of K+channel inhibitors, tetraethylammonium, apamin, dendrotoxin, iberiotoxin, E-4130, chromanol 293B, and tertiapin did not block the epinephrine-induced hyperpolarization. Analysis of whole cell currents revealed an inward conductance of 0.11 ± 0.04 nS/pF ( n = 7) and a TEA-sensitive outward conductance of 0.55 ± 0.08 nS/pF ( n = 7) at -60 and 0 mV, respectively. Guanosine 5′- O-(3-thiotriphosphate) (100 μM) in the patch pipette did not significantly alter these currents or activate novel inward-rectifying K+currents. We conclude that epinephrine can hyperpolarize β-cells in the absence of KATPchannels via activation of low-conductance BaCl2-sensitive K+channels that are regulated by pertussis toxin-sensitive G proteins.