alpha-Ketobutyrate, an intermediate in the catabolism of threonine and methionine, is metabolized to CO2 and propionyl-CoA. Recent studies have suggested that propionyl-CoA may interfere with normal hepatic oxidative metabolism. Based on these observations, the present study examined the effect of alpha-ketobutyrate on palmitic acid and pyruvate metabolism in hepatocytes isolated from fed rats. alpha-Ketobutyrate (10 mM) inhibited the oxidation of palmitic acid by 34%. In the presence of 10 mM carnitine, the inhibition of palmitic acid oxidation by alpha-ketobutyrate was reduced to 21%. These observations are similar to those previously reported using propionate as an inhibitor of fatty acid oxidation, suggesting that propionyl-CoA may be responsible for the inhibition. alpha-Ketobutyrate (10 mM) inhibited 14CO2 generation from [14C]pyruvate by more than 75%. This inhibition was quantitatively larger than seen with equal concentrations of propionate. Carnitine (10 mM) had no effect on the inhibition of pyruvate oxidation by alpha-ketobutyrate despite the generation of large amounts of propionylcarnitine during the incubation. alpha-Ketobutyrate inhibited [14C]glucose formation from [14C]pyruvate by more than 60%. This contrasted to a 30% inhibition caused by propionate. These results suggest that alpha-ketobutyrate inhibits hepatic pyruvate metabolism by a mechanism independent of propionyl-CoA formation. The present study demonstrates that tissue accumulation of alpha-ketobutyrate may lead to disruption of normal cellular metabolism. Additionally, the production of propionyl-CoA from alpha-ketobutyrate is associated with increased generation of propionylcarnitine. These observations provide further evidence that organic acid accumulation associated with a number of disease states may result in interference with normal hepatic metabolism and increased carnitine requirements.