Myoglobin (Mb) has a purported role in facilitating O₂ diffusion in tissue, especially as the cellular PO₂ drops or the respiration demand increases. Inhibiting Mb with CO under conditions that accentuate the facilitated diffusion role should then elicit a significant physiological response. In one set of experiments, the perfused myocardium received buffer with decreasing PO₂ (225, 129, 64 mm Hg). Intracellular PO₂ declined, as reflected in the ¹H NMR Val E11 signal of MbO₂ (67%, 32%, 18%). Adding 6% CO further reduced the available MbO₂ (11%, 9%, 7%), as evidenced by the decline of the MbO₂ Val E11 signal intensity at -2.76 ppm. In a second set of experiments, electrical stimulation increased the heart rate (300, 450, 540 beats per min) and correspondingly the oxygen consumption rate (MVO₂). Intracellular PO₂ also declined, as reflected in the slight drop in the MbO₂ signal (100%, 96%, 82%). MVO₂ increased (100%, 114%, 165%). Adding 3% CO in the stimulated hearts further decreased the available MbO₂ (46%, 44%, 29%). In all cases, CO inactivation of Mb does not induce any change in the respiration rate, contractile function, and high energy phosphate levels. Moreover, the MbCO/MbO₂ partition coefficient shifts dramatically from its in vitro value during hypoxia and increased work and observation suggests a modulation of an intracellular O₂ gradient. Overall, the experimental observations provide then no evidence of a facilitated diffusion role for Mb in perfused myocardium and implicate a physiologically responsive intracellular O₂ gradient.