The intracellular localization of the enzymes in the vitamin B12 dependent pathway which involves the oxidation of propionate was studied in rat liver obtained from normal and vitamin B12 deficient rats as well as from man. The subcellular site of propionyl CoA carboxylase and the vitamin B12 dependent-methylmalonyl CoA mutase were determined. All of the activity of these two enzymes was demonstrated to be in the mitochondria and those enzymes were shown to be loosely bound to the inner membrane-matrix portion of the mitochondria. Vitamin B12 deficiency did not alter the subcellular localization. Finally, a rapid enzymatic assay for methylmalonyl-CoA mutase was described.