It has been proposed that mitochondrial oxidative phosphorylation in pancreatic beta-cells plays an important role in insulin secretion. To examine the impact of mitochondrial dysfunction on insulin secretion, we created a MIN6 cell line that depleted mitochondrial DNA (mtDNA) by treatment with ethidium bromide (EtBr), and studied the response of the cell line to various secretagogues. MIN6 cells cultured with 0.5 microg/ml EtBr for over 2 months (termed MIN6 deltamt cells) revealed a marked (>90%) decrease in mtDNA content and a lack of mRNAs encoded by mtDNA. MIN6 deltamt cells showed the defects of cytochrome c oxidase activity, glucose- and leucine-induced increase in cellular ATP content, and respiratory chain-driven ATP synthesis, suggesting that MIN6 deltamt cells lost oxidative phosphorylation activity due to the selective disruption of the subunits of respiratory chain enzymes encoded by mtDNA. MIN6 deltamt cells also showed a decrease in glucose utilization, suggesting the impairment of the glycolytic pathway as well. After stimulation with glucose and leucine, MIN6 deltamt cells showed no response in insulin secretion or intracellular free Ca2+ concentration ([Ca2+]i). On the other hand, arginine stimulated insulin secretion and an increase in [Ca2+]i in MIN6 deltamt cells as in MIN6 cells. Glibenclamide also stimulated insulin secretion and an increase in [Ca2+]i in both types of cells, but the responses of MIN6 deltamt cells were significantly lower than those of MIN6 cells. These results suggest the importance of ATP production in insulin secretion and an increase in [Ca2+]i, both induced by glucose and leucine. Moreover, mitochondrial function turns out to be not essential but important for the activation of sulfonylurea-induced insulin secretion.