The resting conductance of culturedβ-cells from murine pancreases was investigated using the whole-cell, cell-attached and isolated patch modes of the patch-clamp technique. Whole-cell experiments revealed a high input resistance of the cells (>20 GΩ per cell or>100 kΩ·cm²), if the medium dialysing the cell interior contained 3 mM ATP. The absence of ATP evoked a large additional K⁺ conductance. In cell-attached patches single K⁺-channels were observed in the absence of glucose. Adition of glucose (20 mM) to the bath suppressed the channel activity and initiated action potentials. Similar single-channel currents were recorded from isolated patches. In this case the channels were reversibly blocked by adding ATP (3 mM) to the solution at the intracellular side of the membrane. The conductances (51 pS and 56 pS for [K⁺]₀=145 mM, T=21° C) and kinetics (at −70 mV: τ_open=2.2 ms and τ_closed=0.38 ms and 0.33 ms) of the glucose- and ATP-dependent channels were found to be very similar. It is concluded that both channels are identical. The result suggests that glucose could depolarize theβ-cell by increasing the cytoplasmic concentration of ATP.