Reactive oxygen species (ROS) are believed to be involved in triggering cardiac ischemic preconditioning (IPC). Decreased formation of ROS on reperfusion after prolonged ischemia may in part underlie protection by IPC. In heart models, these contentions have been based either on the effect of ROS scavengers to abrogate IPC-induced preservation or on a measurement of oxidation products on reperfusion. Using spectrophotofluorometry at the left ventricular wall and the fluorescent probe dihydroethidium (DHE), we measured intracellular ROS superoxide (O(2)(-).) continuously in isolated guinea pig heart and tested the effect of IPC and the O(2)(-). scavenger manganese(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) on O(2)(-). formation throughout the phases of preconditioning (PC), 30-min ischemia and 60-min reperfusion (I/R). IPC was evidenced by improved contractile function and reduced infarction; MnTBAP abrogated these effects. Brief PC pulses increased O(2)(-). during the ischemic but not the reperfusion phase. O(2)(-). increased by 35% within 1 min of ischemia, increased further to 95% after 20 min of ischemia, and decreased slowly on reperfusion. In the IPC group, O(2)(-). was not elevated over 35% during index ischemia and was not increased at all on reperfusion; these effects were abrogated by MnTBAP. Our results directly demonstrate how intracellular ROS increase in intact hearts during IPC and I/R and clarify the role of ROS in triggering and mediating IPC.