Reperfusion of the ischemic myocardium leads to a burst of reactive oxygen species (ROS) which is a primary determinant of post-ischemic myocardial dysfunction. We tested the hypothesis that early oxygen delivery and the cellular redox state modulate the initial myocardial ROS production at reperfusion. Isolated buffer perfused rat hearts were loaded with the fluorophores, dihyrofluorescein or Amplex Red to detect intracellular and extracellular ROS formation using surface fluorometry at the left ventricular (LV) wall. Hearts were made globally ischemic for 20 minutes and then reperfused with either 95% or 20% oxygen saturated perfusate. The same protocol was repeated in hearts loaded with dihyrofluorescein and perfused with either 20 mM or 5 mM glucose buffered solution to determine relative changes in NADH and FAD. Myocardial oxygen delivery during the first 5 minutes of reperfusion was 84.7 ± 4.2 ml O₂/min with 20% O₂-saturated buffer and 354.4 ± 22.8 ml O₂/min with 95% O₂ (n=8/group, p<0.001). The fluorescein signal (intracellular ROS) was significantly increased in hearts reperfused with 95% O₂ compared with 20% O2. However, the resorufin signal (extracellular ROS) was significantly increased with 20% O₂ compared with 95% O₂ during reperfusion. Perfusion of hearts with 20 mM glucose reduced the NADH during ischemia (p<0.001) and the ROS at reperfusion (p<0.001) compared with 5.5 mM perfused glucose hearts. Conclusions: Initial oxygen delivery to the ischemic myocardium modulates a compartment-specific ROS response at reperfusion such that high O₂ delivery promotes intracellular ROS and low O₂ delivery promotes extracellular ROS. The redox state which develops during ischemia appears to be an important precursor for reperfusion ROS production.