Disturbances in lipid metabolism may play an important role in the onset of irreversible myocardial damage. To investigate the effect of ischemia and reperfusion on lipid homeostasis and to delineate its possible consequences for myocardial damage, Krebs-Henseleit-perfused, working rat hearts were subjected to various periods of no-flow ischemia (10 to 90 minutes) with or without 30 minutes of reperfusion. During ischemia, the rise in nonesterified fatty acids (NEFAs) was preceded by the accumulation of substantial amounts of glycerol, indicating the presence of an active triacylglycerol-NEFA cycle. The subsequent rise in NEFAs (from 0.25 to 1.64 μmol/g dry residue wt after 90 minutes [means]) coincided with the reduction of ATP to values lower than 10 μmol/g dry wt and the rise of AMP, a potent inhibitor of acyl-coenzyme A synthetase, to values exceeding 2 μmol/g dry wt, making the latter compound a good candidate to hamper the turnover of endogenous lipids during prolonged ischemia. Reperfusion resulted in an additional rise in NEFAs (up to 4.1 μmol/g dry residue wt after 60 minutes of ischemia). Neither ischemia nor reperfusion resulted in significant decreases in the tissue content of triacylglycerols and the various phospholipids. During reperfusion recovery of stroke volume was still adequate at tissue NEFA levels thought to be incompatible with normal mitochondrial function.37A positive correlation (r=0.81) was found between NEFA content of reperfused hearts and cumulative release of lactate dehydrogenase during reperfusion. Accordingly it is concluded that 1) reperfusion results in additional changes in myocardial lipid homeostasis, 2) the accumulating NEFAs are compartmentalized, possibly at the cellular level, and 3) the accumulation of NEFAs is a sensitive marker for myocardial cell damage.