To explore the mechanisms of inhibition of insulin secretion in pancreatic β-cells by oxygen free radicals, we studied the effects of H₂O₂ on membrane currents using the patch-clamp technique. Exposure of β-cells to H₂O₂ (≥30 (μmol/1) increased the activity of ATP-sensitive potassium (K⁺_ATP) channels without changing the single channel conductance in cell-attached membrane patches. Action currents observed during superfusion of 11.1 mmol/1 glucose were suppressed. In inside-out membrane patches, the activity of K⁺_ATP channels was not influenced by H₂O₂. In conventional whole-cell clamp experiments using a pipette solution containing 3 mmol/1 ATP, H₂O₂ did not influence the membrane currents. However, H₂O₂ did activate the K⁺_ATP channel current in perforated whole-cell clamp configurations. The increased K⁺_ATP channel current was reversed by subsequent exposure to 11.1 mmol/12-ketoisocaproic acid. In cell-attached membrane patches, the K⁺_ATP channel current evoked by exposure to 30 μmol/l H₂O₂ was inhibited by exposure to 11.1 mmol/l glyceraldehyde, whereas the channel was again activated by exposure to 0.3 mmol/l H₂O₂. Subsequent superfusion of 11.1 mmol/l 2-ketoisocaproic acid inhibited the channel; this effect was counteracted by exposure to 10 mmol/l H₂O₂. Transient inhibition of K⁺_ATP channels with provocation of action potentials was observed after washout of 100 μmol/l H₂O₂ during superfusion of 2.8 or 11.1 mmol/l glucose. We conclude that H₂O₂ has no direct effect on the K⁺_ATP channels but that it indirectly activates the channels when it is exposed to β-cells under conditions in which the cellular metabolism is physiologically regulated.