The oxygenation state of myoglobin and the redox state of cytochrome c provide information on the PO(2) in the cytosol and mitochondria, respectively. An optical "window" from approximately 540 to 585 nm was found in the pig heart in vivo that permitted the monitoring of myoglobin and cytochrome c without interference from Hb oxygenation or blood volume. Scanning reflectance spectroscopy was performed on the surgically exposed left ventricle of pigs. Difference spectra between control and a total left anterior descending coronary artery occlusion revealed maxima and minima in this spectral region consistent with myoglobin deoxygenation and cytochrome c and b reduction. Comparison of in vivo data with in vitro fractions of the heart, including Hb-free tissue whole heart and homogenates, mitochondria, myoglobin, and pig red blood cells, reveals minimal contributions of Hb in vivo. This conclusion was confirmed by expanding the blood volume of the myocardium and increasing mean Hb O(2) saturation with an intracoronary infusion of adenosine (20 microgram. kg(-1). min(-1)), which had no significant effect on the 540- to 585-nm region. These results also suggested that myoglobin O(2) saturation was not blood flow limited under these conditions in vivo. Work jump studies with phenylephrine also failed to change cytochrome c redox state or myoglobin oxygenation. Computer simulations using recent physical data are consistent with the notion that myoglobin O(2) saturation is >92% under basal conditions and does not change significantly with moderate workloads. These studies show that reflectance spectroscopy can assess myocardial oxygenation in vivo. Myoglobin O(2) saturation is very high and is not labile to moderate changes in cardiac workload in the open-chest pig model. These findings indicate that myoglobin does not contribute significantly to O(2) transport via facilitated diffusion under these conditions.