A new low-field electron paramagnetic resonance approach for noninvasive measurements of myocardial oxygen tension and tissue acidity was developed. The approach was applied to monitor myocardial pO2 and pH in a model of global no-flow ischemia (30 min) and reperfusion in isolated perfused rat hearts. The myocardial oxygen measurements were performed using deuterated Finland trityl radical probe. A rapid decrease in myocardial pO2 from 160 mmHg to about 2 ± 1 mmHg was observed within the first minute of ischemia followed by incomplete restoration of pO2 to 50 mmHg during 30 min of reperfusion. The lower oxygen concentration after ischemia was attributed to the 50% reduction in coronary flow after ischemia as a consequence of myocardial ischemia and reperfusion damage. Myocardial pH measurements using a specially designed imidazoline pH-sensitive nitroxide showed severe myocardial acidification to pH 6.25 during 30 min of ischemia. Preconditioning of the hearts with two 5-min periods of ischemia significantly reduced the acidification of myocardial tissue during sustained ischemia. Noninvasive electron paramagnetic resonance monitoring of myocardial oxygenation and pH may provide important insights into the mechanisms of ischemia and reperfusion injury and a background for development of new therapeutic approaches. Magn Reson Med, 2012. © 2011 Wiley Periodicals, Inc.