Objective: Alterations in myocardial metabolism occur early after the onset of diabetes suggesting that they may play a role in the development of cardiac dysfunction. Inhibition of myocardial pyruvate dehydrogenase (PDH), glucose transport and glycolysis have all been reported following diabetes. In vivo lactate is also a potential source of energy for the heart and its oxidation should not be affected by changes in glucose transport and glycolysis. Therefore, the objective of this study, was to test the hypothesis that following diabetes the inhibition of glucose oxidation would be greater than the inhibition of lactate oxidation. Methods: Hearts from control and one-week-old diabetic rats were perfused with [1-13C]glucose (11 mmol/l) alone, [1-13C]glucose plus lactate (0.5 mmol/l) or glucose plus [3-13C]lactate (0.5 or 1.0 mmol/l) as substrates. Glucose and lactate oxidation rates were determined by combining 13C-NMR glutamate isotopomer analysis of tissue extracts with measurements of oxygen consumption. Results: In diabetic hearts perfused with glucose alone, glucose oxidation was decreased compared to controls (0.31±0.08 vs. 0.71±0.11 μmoles/min/g wet weight; p<0.05). Surprisingly, in hearts perfused with glucose plus 0.5 mmol/l lactate, there was no difference in glucose oxidation between control and diabetic groups (0.20±0.05 vs. 0.16±0.04 μmoles/min/g wet weight respectively). However, under these conditions lactate oxidation was markedly reduced in the diabetic group (0.89±0.18 vs. 0.24±0.05 μmoles/min/g wet weight; p<0.05). At 1.0 mmol/l lactate oxidation was still significantly depressed in the diabetic group. Conclusion: There was a greater decrease in lactate oxidation relative to glucose oxidation in hearts from diabetic animals. These results demonstrate that diabetes leads to a specific inhibition of lactate oxidation independent of its effects on pyruvate dehydrogenase.