Methyl-coenzyme M reductase (MCR), the enzyme responsible for the microbial formation of methane, is a 300-kilodalton protein organized as a hexamer in an alpha(2) beta(2) gamma(2) arrangement. The crystal structure of the enzyme from Methanobacterium thermoautotrophicum, determined at 1.45 angstrom resolution for the inactive enzyme state MCRox1-silent, reveals that two molecules of the nickel porphinoid coenzyme F-430 are embedded between the subunits alpha,alpha’,beta, and gamma and alpha’,alpha,beta’, and gamma’, forming two identical active sites. Each site is accessible for the substrate methyl-coenzyme M through a narrow channel locked after binding of the second substrate coenzyme B. Together with a second structurally characterized enzyme state (MCRsilent) containing the heterodisulfide of coenzymes M and B, a reaction mechanism is proposed that uses a radical intermediate and a nickel organic compound.