Ischemic preconditioning (IPC) strongly protects against ischemia/reperfusion injury, however, its effect on subsequent myocardial oxygenation is unknown. Therefore, we determine in an in vivo mouse model of regional ischemia and reperfusion (I/R) if IPC attenuates postischemic myocardial hyperoxygenation and decreases formation of reactive oxygen/nitrogen species (ROS/RNS), with preservation of mitochondrial function. Five groups of mice: sham, control (I/R), ischemic preconditioning (IPC+I/R, 3 cycles of 5 min coronary occlusion/5 min reperfusion) and IPC+I/R N-nitro-L-arginine methyl ester (L-NAME)-treated, and IPC+I/R eNOS knockout (eNOS^-/-) mice. I/R and IPC+I/R mice were subjected to 30 min regional ischemia followed by 60 min reperfusion. Myocardial PO₂ and redox state were monitored by electron paramagnetic resonance spectroscopy. In IPC+I/R, but not I/R group, regional blood flow was increased after reperfusion. PO₂ upon reperfusion increased significantly above preischemic values in I/R but not in IPC+I/R mice. Tissue redox state was measured from the reduction rate of a spin probe and this rate was 60% higher in IPC than in non-IPC hearts. Activities of NADH dehydrogenase (NADH-DH) and cytochrome c oxidase (CcO) were reduced in I/R mice after 60 min reperfusion but conserved in IPC+I/R mice compared to sham. There were no differences in NADH-DH and CcO expression in I/R and IPC+I/R groups compared to sham. After 60 min reperfusion, strong nitrotyrosine formation was observed in I/R mice but only weak staining in IPC+I/R mice. Thus, IPC markedly attenuates postischemic myocardial hyperoxygenation with less ROS/RNS generation and preservation of mitochondrial O₂ metabolism due to conserved NADH-DH and CcO activities.