Treatment of various types of cells with the mitochondrial ATP-sensitive K+ channel (mK(ATP)) opener has been shown to precondition cells to subsequent injuries and inhibit apoptosis. We exposed cultured osteoblastic MC3T3-E1 cells to hydrogen peroxide (H2O2) with or without pretreatment with a mK(ATP) opener, diazoxide. A marked decrease in osteoblast viability was evident after 48 h exposure of 0.3mM H2O2, compared with vehicle-treated cells. Diazoxide (0.001 approximately 10 microM) treatment significantly (P<0.05) reversed the cytotoxic effect of H2O2 and this effect was blocked by a specific mK(ATP) blocker, glibenclamide. Pretreatment with diazoxide (0.01 approximately 1 microM) also decreased the release of reactive oxygen species and the increase in oxidative damage markers (protein carbonyl and malondialdehyde) induced by H2O2 in osteoblastic MC3T3-E1 cells. Moreover, H2O2-induced reduction of differentiation markers, such as alkaline phosphatase, collagen content and calcium deposition was significantly recovered in the presence of diazoxide. In addition, diazoxide (0.01 approximately 1 microM) decreased the H2O2-induced production of osteoclast differentiation-inducing factors, such as interleukin (IL)-6 and the receptor activator of nuclear factor-kB ligand (RANKL). These results suggest that diazoxide may be useful for the protection of H2O2-induced oxidative damage and dysfunction in osteoblastic cells.