Benzoic acid metabolism, which is primarily a function of liver mitochondria, depending on the concentration of adenosine triphosphate (ATP), coenzyme A (CoA), and glycine in the mitochondrial matrix, was investigated in both rats with long-term cholestasis caused by bile duct ligation (BDL) and sham-operated control rats. In isolated liver mitochondria, hippurate production from benzoate in the presence of saturating glycine concentrations was reduced in BDL rats by 36% with L-glutamate as a source for ATP, by 21% in the presence of succinate, and by 31% in the presence of ATP plus oligomycine. This reduction in benzoate metabolism is in the same range as the previously observed reduction in the activity of the electron transport chain in liver mitochondria from BDL rats. The mitochondrial CoA pool, which can be rate-limiting for benzoic acid metabolism, was not different between BDL and control rats. The activity of benzoyl-CoA synthase, the enzyme catalyzing the rate-limiting step in benzoate metabolism, was reduced by 25%, and the activity of benzoyl-CoA:glycine N-transferase was reduced by 66% in BDL rats. The activity of benzoyl-CoA synthase was significantly inhibited by lithocholate, suggesting that hepatic accumulation of hydrophobic bile acids could contribute to the observed reduction of benzoate metabolism in BDL rats. Benzoate metabolism was also studied in vivo by monitoring the urinary hippurate excretion after intraperitoneal administration of benzoate (100 μmol/100 g of body weight). The time course of hippurate excretion was not different between BDL and control rats. Hippurate excretion over 24 hours after benzoate administration averaged 89.7 ± 4.0% of the administered dose in BDL and 74.4 ± 6.9% (mean ± SEM, difference not significant) in control rats. This finding could be explained by an increase in mitochondrial protein in BDL rats, averaging 2.34 ± 0.29 g per liver in BDL and 1.35 ± 0.07 g per liver in control rats (mean ± SEM, p < .05). Thus, the studies show that benzoate metabolism reflects mitochondrial function in BDL rats both in vivo and in vitro, and that mitochondrial proliferation compensates for the observed decrease in benzoic acid metabolism in isolated mitochondria in vitro.