The contribution of long-chain acyl carnitine to increase enzyme release during ischemia was investigated both in normal and diabetic rat hearts. 2-Tetradecylglycidic acid (TDGA) was used to inhibit acyl carnitine formation. Isolated working-heart preparations were perfused with glucose (11 mM) and palmitate (0.1 mM) in control and mild ischemic conditions. Ischemia induced lactate dehydrogenase (LDH) release from both normal and diabetic hearts, but the release was higher from the diabetics over a 15-min ischemic period. The ischemia-induced tissue accumulation of long-chain acyl carnitine also was greater in diabetic hearts compared with normal hearts. When TDGA was provided in the perfusate 10 min before the addition of palmitate, levels of acyl carnitine were significantly reduced (by approximately 80%) in the ischemic tissue of both groups of hearts. Similarly, LDH release from ischemic hearts was markedly decreased in the presence of TDGA. A positive correlation was shown between LDH release over the ischemic period and the tissue levels of acyl carnitine at the end of ischemia. Significant improvement in mechanical function with TDGA was only observed in ischemic diabetic hearts. There was absolutely no difference in high-energy compounds under a given perfusion condition, either with or without TDGA, between normal and diabetic hearts. It is concluded that lessening the accumulation of fatty acid intermediates, such as acyl carnitine, may be important to prevent or to limit the loss of sarcolemmal integrity under ischemic conditions, especially in diabetic hearts.