To determine whether the effects of fatty acids on the diabetic heart during ischemia involve altered glycolytic ATP and proton production, we measured energetics and intracellular pH (pHi) by using31P NMR spectroscopy plus [2-3H]glucose uptake in isolated rat hearts. Hearts from 7-wk streptozotocin diabetic and control rats, perfused with buffer containing 11 mM glucose, with or without 1.2 mM palmitate or the ketone bodies, 4 mM β-hydroxybutyrate plus 1 mM acetoacetate, were subjected to 32 min of low-flow (0.3 ml · g wet wt−1 · min−1) ischemia, followed by 32 min of reperfusion. In control rat hearts, neither palmitate nor ketone bodies altered the recovery of contractile function. Diabetic rat hearts perfused with glucose alone or with ketone bodies, had functional recoveries 50% lower than those of the control hearts, but palmitate restored recovery to control levels. In a parallel group with the functional recoveries, palmitate prevented the 54% faster loss of ATP in the diabetic, glucose-perfused rat hearts during ischemia, but had no effect on the rate of ATP depletion in control hearts. Palmitate decreased total glucose uptake in control rat hearts during low-flow ischemia, from 106 ± 17 to 52 ± 12 μmol/g wet wt, but did not alter the total glucose uptake in the diabetic rat hearts, which was 42 ± 5 μmol/g wet wt. Recovery of contractile function was unrelated to pHiduring ischemia; the glucose-perfused control and palmitate-perfused diabetic hearts had end-ischemic pHi values that were significantly different at 6.36 ± 0.04 and 6.60 ± 0.02, respectively, but had similar functional recoveries, whereas the glucose-perfused diabetic hearts had significantly lower functional recoveries, but their pHiwas 6.49 ± 0.04. We conclude that fatty acids, but not ketone bodies, protect the diabetic heart by decreasing ATP depletion, with neither having detrimental effects on the normal rat heart during low-flow ischemia.