The quantities of biotinyl proteins in liver of young rats were compared with age-matched controls at intervals during depletion and repletion of biotin. Growth rate and the concentrations of biotinyl proteins previously proposed as mitochondrial storage forms of acetyl CoA carboxylase rapidly decreased in response to biotin deprivation, whereas neither the concentration nor activity of cytosolic acetyl CoA carboxylase was affected. Concentrations of carboxylases active within mitochondria (pyruvate carboxylase, propionyl CoA carboxylase and 3-methyl crotonyl CoA carboxylase) decreased only after d 28. When biotin was injected into biotin-deficient rats, concentrations of the carboxylases active within mitochondria were restored to control levels within 3 h, whereas the concentrations of putative mitochondrial storage forms of acetyl CoA carboxylase reached normal levels only after 9 h, indicating that the injected biotin was preferentially used for the synthesis of the carboxylases active within mitochondria rather than acetyl CoA carboxylase. Mitochondrial acetyl CoA carboxylase may serve as a reservoir to maintain a normal concentration of cytosolic acetyl CoA carboxylase in liver of rats deprived of biotin and provide biotin, indirectly, to maintain essentially normal concentrations of the biotinyl enzymes active within mitochondria for several weeks after rats were fed a biotin-deficient diet.