The time course of changes in the myocardial levels of nonesterified fatty acids (NEFA), adenosine triphosphate (ATP), creatine phosphate (CrP) and lactate, and those in the cardiac mechanical function during ischemia and reperfusion was investigated in the isolated, perfused, working rat heart. Ischemia was produced by lowering the afterload pressure from 60 to 0 mm Hg, and reperfusion resulted from raising the afterload pressure to 60 mm Hg. Ischemia stopped the heart beat, and increased the myocardial levels of unsaturated NEFA (such as arachidonic, palmitoleic, and linoleic acids) as a function of the ischemic period; it decreased the myocardial levels of ATP and CrP, and increased the myocardial level of lactate. The level of arachidonic acid increased when the myocardial level of ATP fell below 5 mumol/g dry weight. Reperfusion after ischemia started the heart beat, and restored the mechanical function which depended on the preceding ischemic period. Reperfusion also increased the levels of ATP and CrP and decreased the level of lactate, whereas it further increased the levels of the NEFA that had been elevated by ischemia. The recovery of mechanical function was inversely correlated with the myocardial level of arachidonic acid during ischemia and reperfusion. We concluded that changes in the myocardial levels of NEFA during ischemia and reperfusion are different from those of ATP, CrP, and lactate, and suggest that the myocardial level of arachidonic acid during ischemia and reperfusion can be a sensitive and suitable marker for the recovery of mechanical function during reperfusion.