Long-term caloric restriction (CR) has been shown to extend maximum life span in laboratory rodents. We investigated the activities of gluconeogenic and transaminase enzymes in the livers of old and young mice fed either control or calorie-restricted diets. Livers were sampled 48 h after the last scheduled feeding time. Old mice on CR showed significant increases in the activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase when compared with controls, indicating increased gluconeogenesis. Increased activities of tyrosine, tryptophan, histidine, phenylalanine, alanine and aspartate transaminases, as well as of malate and glutamate dehydrogenases were also observed, while branched-chain amino acid transaminase was unchanged. Young mice on CR showed a significant increase only in the phosphoenolpyruvate carboxykinase activity in the gluconeogenic pathway, while transaminases were increased significantly, except for tryptophan and branched-chain amino acid transaminases. Glutamate dehydrogenase also showed increased activity but malate dehydrogenase was unchanged. Increases in the level of acetyl-CoA and [Acetyl-CoA]/[CoA] ratio were observed only in the old CR mice. Our results demonstrate increased gluconeogenic activity in CR mice and are consistent with a state of increased hepatic gluconeogenesis and protein turnover during CR.