In pancreatic B-cells insulin hyperpolarises the plasma membrane (Vm) via activation of KATP channels. We investigated whether insulin affects B-cell function by KATP channel-independent mechanisms. SUR1-KO B-cells responded to an increase in glucose concentration (0.5 to 15 mM) with a hyperpolarisation of Vm (-8±1 mV, n=6) and a decrease of [Ca2+ ]c that did not result from Ca2+ uptake into the ER (n=7). A similar glucose effect was obtained in WT B-cells when KATP channels were blocked by tolbutamide (n=4). In SUR1-KO B-cells the reduction of [Ca2+ ]c was attenuated by the tyrosine kinase blocker genistein and the PI3 kinase inhibitor wortmannin suggesting that it is mediated by glucose-stimulated insulin release. 200?400 nM insulin hyperpolarised Vm by -4±1 mV (n=4) and changed Ca2+ oscillations. Elevation of glucose concentration as well as application of insulin reduced the intracellular Na+ concentration (n=4-9). The Na,K-ATPase inhibitor ouabain prevented the effect of 15 mM glucose on Vm, [Ca2+ ]c and [Na+ ]c (n=3-5). These data suggest that the insulin-induced membrane hyperpolarisation involves activation of the Na,K-ATPase and that this mechanism influences B-cell function in patients treated with sulfonylureas.