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.