In ischemic preconditioning (IPC) brief ischemia/reperfusion renders the heart resistant to infarction from any subsequent ischemic insult. Protection results from binding of surface receptors by ligands released during the preconditioning ischemia. The downstream pathway involves redox signaling as IPC will not protect in the presence of a free radical scavenger. To determine when in the IPC protocol the redox signaling occurs, seven groups of isolated rabbit hearts were studied. All hearts underwent 30 min of coronary branch occlusion and 2 h of reperfusion. IPC groups were subjected to 5 min of regional ischemia followed by 10 min of reperfusion prior to the 30-min coronary occlusion. The Control group had only the 30-min occlusion and 2-h reperfusion. In the second group IPC preceded the index coronary occlusion. The third group was also preconditioned, but the free radical scavenger N-2-mercaptopropionyl glycine (MPG 300 µM) was infused during the 10-min reperfusion and therefore was present in the myocardium in the distribution of the snared coronary artery during the entire reperfusion phase and also during the subsequent 30-min ischemia. In another preconditioned group MPG was added to the perfusate before the preconditioning ischemia and therefore was present in the tissue only during the preconditioning ischemia and then was washed out during reperfusion. In the fifth group MPG was added to the perfusate for only the last 5 min of the preconditioning reperfusion and therefore was present in the tissue during the last minutes of the reperfusion phase and the 30 min of ischemia. In an additional group of IPC hearts MPG was infused for only the initial 5 min of the preconditioning reperfusion and then allowed to wash out so that the scavenger was present for only the first half of the reperfusion phase. Infarct and risk zone sizes were measured by triphenyltetrazolium staining and fluorescent microspheres, resp. IPC reduced infarct size from 31.3 ± 2.7% of the ischemic zone in control hearts to only 8.4 ± 1.9%. MPG completely blocked IPC’s protection in the third (39.4 ± 2.8%) and sixth (36.1 ± 7.7%) groups but did not affect its protection in groups 4 (8.1 ± 1.5%) or 5 (7.8 ± 1.1%). When deoxygenated buffer was used during IPC’s reperfusion phase in the seventh group of hearts, protection was lost and infarct size was increased over that seen in control hearts (74.5 ± 9.0%). Hence redox signaling occurs during the reperfusion phase of IPC, and the critical component in that reperfusion phase appears to be molecular oxygen.