The physical law of diffusion imposes O₂ concentration gradients from the plasma membrane to the center of cell. The present study was undertaken to determine how such intracellular radial gradients of O₂ affect the fate of isolated single cardiomyocytes. In single rat cardiomyocytes, mitochondrial respiration was moderately elevated by an oxidative phosphorylation uncoupler to augment the intracellular O₂ gradient. At physiological extracellular O₂ levels (2-5%), decreases in myoglobin O₂ saturation and increases in NADH fluorescence at the center of the cell were imaged (anoxic cell core), while the mitochondrial membrane potential (_m) and ATP levels at the anoxic cell core were relatively sustained. In contrast, treatment with 0.5 mM iodoacetamide (IA) to inhibit creatine kinase (CK) resulted in depletion of both _m and ATP at the anoxic cell core. Even at normal extracellular Po₂, actively respiring cardiomyocytes developed rigor contracture followed by necrotic cell death. Furthermore, such rigor was remarkably accelerated by IA, while cell injury was perfectly rescued by mitochondrial F₁F_o inhibition by oligomycin. These results suggest that increases in radial gradients of O₂ potentially promote cell death through the reverse action of F₁F_o in mitochondria located at the anoxic cell core. However, in the intact cardiomyocyte, the CK-mediated energy flux from the subsarcolemmal space may sustain _m at the cell core, thus avoiding uncontrolled consumption of ATP that can lead to necrotic cell death. Mitochondria at the anoxic core can cause necrotic cell death in cardiomyocytes at physiological extracellular Po₂.