Oxygen-free radicals are thought to be a major cause of β-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H₂O₂ on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the activity of ATP-sensitive potassium (K⁺_ATP) channels in isolated rat pancreatic β-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H₂O₂ in the presence of 2.8 mM glucose increased [Ca²⁺]_i from 114.3±15.4 nM to 531.1±71.9 nM (n=6) and also increased frequency of K⁺_ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H₂O₂ inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H₂O₂, followed by a transient increase in [Ca²⁺]_i, the transient inhibition of K⁺_ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca²⁺, 0.1 mM H₂O₂ increased [Ca²⁺]_i from 88.8±7.2 nM to 134.6±8.3 nM. Magnitude of [Ca²⁺]_i increase induced by 0.1 mM H₂O ₂ was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺ pump (45.8±4.9 nM vs 15.0±4.8 nM). Small increase in [Ca²⁺]_i in response to an increase of external Ca²⁺ from zero to 2 mM was further facilitated by 0.1 mM H₂O₂ (330.5 ±122.7 nM). We concluded that H₂O₂ not only activates K⁺_ATP channels in association with metabolic inhibition, but also increases partly the Ca²⁺ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic β-cells.