Saturation transfer from gamma-ATP to inorganic phosphate was used to assign the intracellular inorganic phosphate resonance of the phosphorus-31 nuclear magnetic resonance spectrum of heart obtained from adult sheep under Halothane anesthesia. The 31P chemical shift of intracellular inorganic phosphate was then used as a probe of myocardial pH. Resting myocardial pH was found to be 7.03 +/- 0.02. The effects of increasing myocardial work on myocardial pH were examined using external pacing and phenylephrine infusion alone or in combination to produce steady-state increases in the rate-pressure product. No alteration in myocardial pH was observed with up to 4-fold increases in rate-pressure product. No changes in high-energy phosphates were observed except at the highest rate-pressure products obtained, where small increases in inorganic phosphate and decreases in the phosphocreatine/ATP ratio were observed. In addition, the transition to a new steady state was studied with a 20-s time resolution after initiation of pacing. Again, no changes in pH or levels of phosphates were detected during the transition to increased work.