Two enzymes involved in the intracellular metabolism of cobalamin have been identified and characterized: cyanocobalamin beta-ligand transferase and microsomal cob(III)alamin reductase. The beta-ligand transferase is a cytosolic enzyme utilizing FAD, NADPH and reduced glutathione. The product of the reaction has been identified as glutathionyl-cobalamin. NADH-linked cob(III)alamin reductase has been found in two subcellular fractions: microsomal and inner mitochondrial membrane. The product of the reduction catalyzed by the microsomal enzyme has been identified as cob(II)alamin. In cbl C mutant fibroblasts, the specific activities of cyanocobalamin beta-ligand transferase and cob(III)alamin reductase were markedly decreased and have varied from 3%-30% and 36%-42% of normal, respectively. The specific activity of mitochondrial cob(III)alamin reductase was only 30% of normal in two cbl C mutants and normal in remaining mutant cell lines. In the cbl D cells, the specific activities were 33% and 55%. Mitochondrial cob(III)alamin reductase was not affected by cbl D mutation. Methionine synthase, L-methylmalonyl-CoA mutase and microsomal cytochrome c and b5 reductases are not affected by both mutations. The cbl E mutation affects only the activity of methionine synthase. These results support the hypothesis that the early enzymatic steps of intracellular metabolism of cobalamin are similar in the synthesis of both methylcobalamin and adenosylcobalamin and these steps are altered by the cbl C and cbl D mutations.