Leucine metabolism in skeletal muscle is linked to protein turnover. Since clofibrate is known both to cause myopathy and to decrease muscle protein content, the present investigations were designed to examine the effects of acute clofibrate treatment on leucine oxidation. Rat skeletal muscle cells in tissue culture were used in these studies because cultivated skeletal muscle cells, like muscle in vivo, have been shown to actively utilize branched chain amino acids and to produce alanine. The conversion of [1-(14)C]leucine to (14)CO(2) or to the [1-(14)C]keto-acid of leucine (alpha-keto-isocaproate) was linear for at least 2 h of incubation; the production of (14)CO(2) from [1-(14)C]leucine was saturable with a K(m) = 6.3 mM and a maximum oxidation rate (V(max)) = 31 nmol/mg protein per 120 min. Clofibric acid selectively inhibited the oxidation of [1-(14)C]leucine (K(i) = 0.85 mM) and [U-(14)C]isoleucine, but had no effect on the oxidation of [U-(14)C]glutamate, -alanine, -lactate, or -palmitate. The inhibition of [1-(14)C]leucine oxidation by clofibrate was also observed in the rat quarter-diaphragm preparation. Clofibrate primarily inhibited the production of (14)CO(2) and had relatively little effect on the production of [1-(14)C]keto-acid of leucine. A physiological concentration-3.0 g/100 ml-of albumin, which actively binds clofibric acid, inhibited but did not abolish the effects of a 2-mM concentration of clofibric acid on leucine oxidation. Clofibrate treatment stimulated the net consumption of pyruvate, and inhibited the net production of alanine. The drug also increased the cytosolic NADH/NAD(+) ratio as reflected by an increase in the lactate/pyruvate ratio, in association with a decrease in cell aspartate levels. The changes in pyruvate metabolism and cell redox state induced by the drug were delayed compared with the nearly immediate inhibition of leucine oxidation. These studies suggest that clofibric acid, in concentrations that approximate high therapeutic levels of the drug, selectively inhibits branched chain amino acid oxidation, possibly at the level of the branched chain keto-acid dehydrogenase.