Oxygen-free radicals are thought to be a major cause of b-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 b-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 H2O2 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 b-cells.