The role of mitochondrial K(ATP) (mitoK(ATP)) channels on muscle fatigue was assessed in adult mouse skeletal muscle bundles. Muscle fatigue was produced by eliciting short repetitive tetani. Isometric tension and the rate of production of reactive oxygen species (ROS) were measured at room temperature (20-22 degrees C) using a force transducer and the fluorescent indicator CM-H(2)DCFDA. We found that opening mitoK(ATP) channels with diazoxide (100 microM) significantly reduced muscle fatigue. Fatigue tension was 34% higher in diazoxide-treated fibers relative to controls. This effect was blocked by the mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD), by the protein kinase C (PKC) inhibitor chelerythrine, and by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) but was not accompanied by a change in the rate of ROS production during fatigue. A physiological role of mitoK(ATP) channels on muscle fatigue is proposed.