Myoglobin (Mb) has a purported role in facilitating O2 diffusion in tissue, especially as 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, and 64 mmHg). Intracellular PO2 declined, as reflected in the 1H NMR Val E11 signal of MbO2 (67%, 32%, and 18%). The addition of 6% CO further reduced the available MbO2 (11%, 9%, and 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, and 540 beats/min) and correspondingly the O2 consumption rate (MVO2). Intracellular PO2 also declined, as reflected in the slight drop in the MbO2 signal (100%, 96%, and 82%). MVO2 increased (100%, 114%, 165%). The addition of 3% CO in the stimulated hearts further decreased the available MbO2 (46%, 44%, and 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. The observation suggests a modulation of an intracellular O2 gradient. Overall, the experimental observations provide no evidence of a facilitated diffusion role for Mb in perfused myocardium and implicate a physiologically responsive intracellular O2 gradient.