The activities of key gluconeogenic enzymes in livers of biotin-deficient rats under fed and fasting condition were examined. PEP carboxykinase, FDPase and G6Pase increased significantly on fasting and were not affected by the deficiency. There was a 10- to 20-fold decrease in pyruvate carboxylase activity in the deficiency and fasting had no effect on the activity. Within 1 hour after the administration of biotin to deficient animals there was a 5-fold increase in pyruvate carboxylase activity. Since actinomycin D or puromycin had no effect on the restoration of the activity invivo it appears that pyruvate apocarboxylase and the holoenzyme synthetase were present in the deficient liver and once biotin was available, invivo synthesis of pyruvate holocarboxylase occurred. © 1969.