Hepatic encephalopathy is a severe neuropathological condition arising secondary to liver failure. The pathogenesis is not well understood; however, hyperammonemia is considered to be one causative factor. Hyperammonemia has been suggested to inhibit tricarboxylic acid (TCA) cycle activity, thus affecting energy metabolism. Furthermore, it has been suggested that catabolism of the branched-chain amino acid isoleucine may help curb the effect of hyperammonemia by by-passing the TCA cycle block as well as providing the carbon skeleton for glutamate and glutamine synthesis thus fixating ammonia. Here we present novel results describing [U-(13)C]isoleucine metabolism in muscle and brain analyzed by mass spectrometry in bile duct ligated rats, a model of chronic hepatic encephalopathy, and discuss them in relation to previously published results from neural cell cultures. The metabolism of [U-(13)C]isoleucine in muscle tissue was about five times higher than that in the brain which, in turn, was lower than in corresponding cell cultures. However, synthesis of glutamate and glutamine was supported by catabolism of isoleucine. In rat brain, differential labeling patterns in glutamate and glutamine suggest that isoleucine may primarily be metabolized in the astrocytic compartment which is in accordance with previous findings in neural cell cultures. Lastly, in rat brain the labeling patterns of glutamate, aspartate and GABA do not suggest any significant inhibition by ammonia of TCA cycle activity which corresponds well to findings in neural cell cultures. Branched-chain amino acids including isoleucine are used for treating hepatic encephalopathy and the present findings shed light on the possible mechanism involved. The low turn-over of isoleucine in rat brain suggests that this amino acid does not serve the role of providing metabolites pertinent to TCA cycle function and hence energy formation as well as the necessary carbon skeleton for subsequent ammonia fixation in hyperammonemia. The higher metabolism of isoleucine in muscle could, however, contribute to ammonia fixation and thus likely be of value in the treatment of hepatic encephalopathy.