N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane associated, corrinoid-containing enzyme complex which uses a methyl-transfer reaction to drive an energy-conserving sodium ion pump. The purified methyltransferase from Methanobacterium thermoautotrophicum (strain Marburg) exhibited a rhombic EPR signal indicative of a base-on cob(II)amide. In this form, the enzyme was almost completely inactive. Upon addition of Ti(III)citrate, which is a one-electron reductant known to reduce corrinoids to the cob(I)amide form, the EPR signal was completely quenched. In the reduced form, the enzyme was active. When the purified complex was incubated in the presence of both Ti(III) and N5-methyltetrahydromethanopterin (CH3-H4MPT), enzyme-bound Co-methyl-5'-hydroxybenzimidazolyl cob(III)amide was formed. Upon incubation of the methylated enzyme with either tetrahydromethanopterin or coenzyme M, the enzyme was demethylated with the concomitant formation of CH3-H4MPT and methylcoenzyme M, respectively. Enzyme demethylation, in contrast to enzyme methylation, was not dependent on the presence of Ti(III). Methyl transfer from the methylated enzyme to coenzyme M was essentially irreversible. These results are interpreted to that the purified enzyme complex is active only when the enzyme-bound corrinoid is in the reduced cob(I)amide form, and that methyl transfer from CH3-H4MPT to coenzyme M proceeds via nucleophilic attack of the cobalt(I) on the N5-methyl substituent of CH3-H4MPT, forming an enzyme-bound CH3-corrinoid as intermediate. Methyl-coenzyme M formation from CH3-H4MPT and coenzyme M, as catalyzed by the purified methyltransferase, was stimulated by sodium ions, half-maximal activity being obtained at approximately 50 microM Na+. We therefore infer that the methyltransferase, as isolated, is capable of vectorial sodium ion translocation.