We investigated whether the antiepileptic valproic acid (VPA) might interfere with oxidative metabolism in heart, as it does in liver. We administered VPA to working rat hearts perfused with radiolabeled carbohydrate and fatty acid fuels. Measurements included oxidation rates of (i) glucose, pyruvate, or lactate in the presence of palmitate and (ii) palmitate, octanoate, or butyrate in the presence of glucose. Oxidation rates were quantified as the rate of appearance of 14CO2 or 3H2O from 14C- or 3H-labeled substrates. In hearts perfused with palmitate, VPA (1 mmol/L) strongly inhibited the oxidation of pyruvate and lactate but slightly stimulated the oxidation of glucose. VPA also inhibited lactate or pyruvate uptake into erythrocytes in vitro. In hearts perfused with glucose, VPA strongly inhibited the oxidation of palmitate and octanoate but had no effect on butyrate oxidation. The absence of valproate CoA ligase activity in cell-free homogenates indicated that the inhibition of fatty acid oxidation by VPA did not require prior activation to valproyl-CoA. The results are consistent with the hypothesis that VPA selectively interferes with myocardial fuel oxidation by mechanisms that are independent of conversion to the CoA thioester.