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Pyruvate carboxylase purified from calf liver contains bound magnesium and bound manganese in a combined stoichiometry equivalent to the biotin content of this enzyme. Both metal ions are well correlated with enzymic activity when this enzyme is fractionated on columns of DEAE-Sephadex A-50 (SO42-) or Sephadex G-200. The bound metal status of this mammalian liver pyruvate carboxylase, therefore, differs from the pyruvate carboxylases purified from chicken (Scrutton, M. C., and Mildvan, A. S. (1968) Biochemistry 7, 1940) and turkey liver which are identified as manganese metallo-biotin enzymes. Magnesium replaces manganese as the bound metal of pyruvate carboxylase purified from the livers of chickens which have been raised on rations deficient in manganese. The relative content of magnesium and manganese in the purified enzyme reflects the manganese content of the ration on which the chickens were raised. When the ration containing the lowest level of manganese (0.3 mg per kg) is fed, the pyruvate carboxylase obtained is a magnesium metallobiotin enzyme. Substitution of magnesium for manganese occurs with retention of catalytic activity and causes only minor alterations in the catalytic properties of pyruvate carboxylase. Properties which are affected by the substitution include the apparent Km for pyruvate and the concentration of oxalate required for 50% inhibition of the over-all reaction. In the case of inhibition by oxalate the effect observed is in accord with predictions based on the properties of model complexes.