The potential role of phosphorylated compounds in the control of myocardial cell respiration was investigated by means of 31P-nuclear magnetic resonance (NMR) spectroscopy. Isolated isovolumic rat hearts, perfused with a 9 mM glucose, 2 mM pyruvate medium at a constant beating rate (6 Hz) and temperature (37 degrees C), were subjected to changes in work load by varying the calcium concentration ([Ca2+]) in the perfusion fluid from 0.5 to 1.0, 1.5, or 2.0 mM. Each change in left ventricular developed pressure (LVDP) induced by the [Ca2+] change was accompanied by alterations in the inorganic phosphate-to-creatine phosphate ratio ([Pi]/[PCr]), with the ATP level remaining constant. The relationship between [Pi]/[PCr] and LVDP followed a Michaelis-Menten pattern with an apparent Michaelis constant (Km) of 0.09 and a maximal LVDP of 91 mmHg. This Km corresponded to intracellular concentrations of 1.2 mM for Pi and 13.0 mM for PCr. The calculated [ADP] and phosphorylation potential corresponding to these values were 44 microM and 151,000 M-1, respectively. All these values are close to those estimated under in situ physiological conditions. These results support the assumption that in the rat heart, as in skeletal muscle, mitochondrial activity could be controlled by changes in phosphorylated compound concentrations under normoxic conditions.