Diabetic hearts are reported to be more dependent on fatty acids (FA) for energy production, despite observations that FA oxidation is reduced. These conclusions are based primarily on work using only one concentration of FA with glucose as the sole additional substrate. We studied the effect of increasing palmitate (P) concentration on lactate (L) and P oxidation in hearts from control and diabetic rats, perfused with glucose (5 mM) + [3-13C]L (0.5 mM) + [U-13C]P (0, 0.1, 0.32 or 1 mM) as substrates. The relative contributions of L and P to the TCA cycle were calculated from high resolution 13C-NMR spectra of tissue extracts (See Figure). At 0.1mM P, L oxidation decreased by <10% in controls relative to 0mM; however, there was a 50% decrease in the diabetic group. At 1.0 mM P there was no difference in L oxidation between the two groups. The contribution of P to the TCA cycle was linear in both groups, but the slopes were markedly different. At 0.1 mM P, the contribution of P to the TCA cycle in diabetics was significantly higher than controls; however, at 1.0mM P it was significantly lower. In summary, L oxidation in diabetic hearts appears to be more sensitive to inhibition by P compared to controls, although at 1mM P, L oxidation is similar in both groups. The contribution of P to the TCA cycle is linearly related to the exogenous palmitate concentration in both groups; however the slope of this relationship is clearly decreased following diabetes.