The ability to measure ATP synthesis rates using 31P‐NMR spectroscopy is demonstrated in the normal, ischemic, and postischemic myocardium in vivo. Cardiopulmonary bypass (CBP) was employed to induce 20 min of global myocardial ischemia, and to conduct magnetization transfer measurements during the ischemic episode and following reperfusion and return to normal circulation. For the first few minutes of ischemia, transfer of magnetization from ATPγ to P1 was extensive and the resultant fractional reduction (ΔM/Mo) in the Pi resonance intensity reached approximately 100%. Subsequent to reperfusion and stabilization off CPB and on normal circulation, both the fractional reduction and the spin‐lattice relaxation time, T 1*, of the Pi resonance were determined when ATP, spins were saturated. Under these conditions, the unidirectional ATP synthesis rate was 0.41 ± 0.09 (SEM, N = 4) μmol/s/g wet wt. The data suggest that in the canine myocardium in vivo, glycolytic enzymes mediate a very rapid exchange between Pi and ATP γ‐phosphates during early phases of ischemia; in the postischemic reperfused myocardium, however, the glycolytic contribution to the unidirectional Pi → ATP rate measured by NMR in vivo is relatively small compared to that observed in glucose‐perfused, postischemic rat hearts. © 1990 Academic Press, Inc.