The purpose of the present study was to visualize myoglobin-facilitated oxygen delivery to mitochondria at a critical mitochondrial oxygen supply in single isolated cardiomyocytes of rats. Using the autofluorescence of mitochondrial reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), the mitochondrial oxygen supply was imaged from approximately 1.4 microm inside the cell surface at a subcellular spatial resolution. Significant radial gradients of intracellular oxygenation were produced by superfusing the cell suspension with a mixed gas containing 2-4% oxygen while stimulating mitochondrial respiration with an uncoupler of oxidative phosphorylation. Augmentation of the NAD(P)H fluorescence started from the core of the cell (anoxic core) and progressively expanded toward the plasma membrane, as the extracellular Po(2) was lowered. Inactivation of cytosolic myoglobin by 5 mM NaNO(2) significantly enlarged such anoxic regions. Nitrite affected neither mitochondrial respiration in uncoupled cells nor the relationship between Po(2) and the NAD(P)H fluorescence in coupled cells. Thus we conclude that myoglobin significantly facilitates intracellular oxygen transport at a critical level of mitochondrial oxygen supply in single cardiomyocytes.