The overall activity and distribution of pyruvate carboxylase and of phosphoenolpyruvate carboxykinase in rat liver was studied by applying an improved technique for the fractional extraction of hepatic enzymes. In the rat the mean activity of pyruvate carboxylase amounted to 10 units/g fresh liver (1 unit = 1 μmole pyruvate carboxylated/min at 30°). In the human liver the corresponding value was about 1 unit/g fresh wt. For phosphoenolpyruvate carboxy-kinase 2.5 and 10 units/g fresh wt. were measured in rat and human liver, respectively. During fractional extraction only negligible quantities of pyruvate carboxylase appeared in the soluble cytoplasmatic fraction. It is concluded that the bulk of enzyme activity is located within the mitochondria. By digitonin treatment of isolated rat liver mitochondria pyruvate carboxylase could be localized within the matrix space. Phosphoenolpyruvate carboxykinase was found to be located mainly in the soluble cytoplasmatic fraction. Our results strongly support the view that in rat liver gluconeogenesis pyruvate has to enter the mitochondria prior to its carboxy-lation to oxaloacetate.