Mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels have been suggested as triggers and end effectors in myocardial ischemic preconditioning. However, the intracellular mechanism regulating mitoK(ATP) channels remains unclear. In the present study, mitoK(ATP) channels from bovine ventricular myocardium were reconstituted using planar lipid bilayers, and the effect of superoxide (O(2-.)) on the activity of these reconstituted channels was examined. After incorporation, a potassium-selective current was recorded. The mean conductance of this current was 56 pS at 150 mmol/L KCl, which was substantially inhibited by 1 mmol/L MgATP. 5-Hydroxydecanoate (5-HD, 10 to 100 micromol/L), a selective mitoK(ATP) antagonist, reduced the open state probability (NPo) of these channels in a concentration-dependent manner, whereas diazoxide (10 micromol/L), a selective mitoK(ATP) agonist, significantly increased channel activity. HMR-1098 (100 micromol/L), a selective sarcolemmal K(ATP) antagonist, had no effect on the activity of reconstituted channels. Addition of xanthine/xanthine oxidase (100 micromol/L per 0.038 U/mL), an O(2-.)-generating system, resulted in a marked activation of mitoK(ATP) channels; the NPo of the channels was increased from 0.60+/-0.10 to 1.94+/-0.02. This O(2)(-.)-induced channel activation was completely abolished by pretreatment with 5-HD (100 micromol/L) or a sulfhydryl alkylating compound, N-ethylmaleimide (2 mmol/L). It is concluded that myocardial mitoK(ATP) channels can be reconstituted into lipid bilayers and that O(2-.) activates these channels. The effect of O(2-.) may be associated with its direct action on the sulfhydryl groups of the channel protein.