The effect of supplying exogenous methylcobalamin (MeCbl), a methyl donor to methionine synthase (MS), on the cellular metabolism of MeCbl was tested in cultured fibroblasts from healthy persons and from a subject with an inherited defect in the synthesis of MeCbl. MeCbl bound to transcobalamin II (TCII) was taken up in larger amounts than cyanocobalamin (CN-Cbl), but was equal to the uptake of hydroxocobalamin (OH-Cbl). The form of Cbl in the lysosomes persisted as the same form, bound to TCII, to which the cells were exposed in the medium. Once released from the lysosomes, both MeCbl and OH-Cbl were converted in the same proportions to coenzyme forms, suggesting equivalent entry into common cellular pools of Cbl from which active forms are synthesized. Exogenous MeCbl enjoyed no advantage in binding to MS, in synthesis of MeCbl, and in supporting cell division in the absence of methionine. All evidence supported the concept that in human cells the active MeCbl on MS forms de novo on the enzyme. It appeared unlikely that therapeutic MeCbl would have any advantage over OH-Cbl in the treatment of MeCbl deficiency or Cbl deficiency in general.