Conventional electrophysiology and the whole-cell patch-clamp technique have been applied to elucidate the effects of H₂O₂ on pancreatic B-cells of the mouse. In these cells, addition of 15 mmol/l glucose leads to depolarization and oscillation of the cell membrane potential. Subsequent addition of H₂O₂ (1 mmol/l) in the presence of glucose was followed by a marked and rapid hyperpolarization of the cell membrane with suppression of the electrical activity. Accordingly, in slow whole-cell patch-clamp experiments (with nystatin in the pipette solution) H₂O₂ induced a marked increase of cell membrane conductance. Tolbutamide, a blocker of K⁺ _ATP channels, only partially blocked the effect of H₂O₂ even at high concentrations. The H₂O₂-induced, tolbutamide-insensitive current component, however, was largely abolished by a high concentration of TEA⁺ (80 mmol/l) or BaCl₂ (10 mmol/l). It is concluded that in B-cells H₂O₂ stimulates a K⁺ current and that this effect leads to marked hyperpolarization and reversal of glucose-induced oscillations of cell membrane potential.