Because the net Ca²⁺ uptake in the sarcoplasmic reticulum (SR) of cardiac muscle is a result of the activity of Ca²⁺-ATPase and of the SR Ca²⁺-release channel, an abnormal Ca²⁺ uptake may be the result of the dysfunction of either or both structures. The site or sites of action for oxygen-derived free radicals (OFR) damage are unknow, although previous studies on the SR have focused on damage to the Ca²⁺ pump. Direct effects of OFR on SR Ca²⁺-release channels may be important in understanding their potential contribution to myocardial ischemia/reperfusion injury. We confirmed that superoxide anion radical (O2^·-) generated from hypoxanthine-xanthine oxidase reaction decreases calmodulin content and increases ⁴⁵Ca²⁺ efflux from the heavy fraction of canine cardiac SR vesicles. Electron spin resonance study showed that hydroxyl radicals are generated in addition to O2^·- from hypoxanthine-xanthine oxidase reaction, and data indicate that O2^·- is responsibe for the observed effect. Current fluctuations through single Ca²⁺-release channels have been also monitored after incorporation into planar phospholipid bilayers. We directly demonstrate that activation of the channel by O2^·- stimulates Ca²⁺release from heavy SR vesicles and suggest the importance of accessory proteins such as calmodulin in modulating the effect of O2^·-.