Methanococcus voltae carries genetic information for four [NiFe] hydrogenases. Two of the hydrogenases are predicted to contain selenocysteine on the basis of in-frame TGA codons, while the genes encoding the two other enzymes contain cysteine codons at homologous positions. Their predicted subunit compositions and their electron acceptor specificities are similar to those of the respective selenium-containing enzymes. The selenium-containing hydrogenases have been purified and characterized. Only one of them reduces the deazaflavin F(420). The activity of the F(420)-nonreducing enzyme is exceptionally high. The selenium atom has been shown by EPR spectroscopy to be a ligand to the Ni atom in the primary reaction centers in both enzymes. The spectroscopic analyses also yielded a description of the electronic configuration around the NiFe center at different oxidation states and in the presence of the competitive inhibitor, CO. The genes encoding the selenium-free hydrogenases are expressed only in the absence of selenium. They are linked by an intergenic region in which regulatory cis elements were defined by employing reporter gene constructs and site-directed mutagenesis.