[NiFeSe] hydrogenases are a subgroup of the large family of [NiFe] hydrogenases in which a selenocysteine ligand coordinates the Ni atom at the active site. As observed for other selenoproteins, the [NiFeSe] hydrogenases display much higher catalytic activities than their Cys-containing homologues. Here, we review the biochemical, catalytic, spectroscopic and structural properties of known [NiFeSe] hydrogenases, namely from the Hys (group 1 [NiFeSe] hydrogenase), Fru (F-420-reducing [NiFeSe] hydrogenases) and Vhu families (F-420-non-reducing [NiFeSe] hydrogenases). A survey of new [NiFeSe] hydrogenases present in the databases showed that all enzymes belong to either group 1 periplasmic uptake hydrogenases (Hys) or to group 3 cytoplasmic hydrogenases (Fru and Vhu) and are present in either sulfate-reducing or methanogenic microorganisms. In both kinds of organisms, the [NiFeSe] hydrogenases are preferred over their Cys-containing homologues if selenium is available. Since no structural information is available for the Vhu and Fru enzymes, we have modelled the large subunit of these enzymes and analyzed the area surrounding the active site. Three [NiFeSe] hydrogenases of the Hys and Vhu types were identified in which the selenocysteine residue is found in a different location in the sequence, which could result in a different coordination to the Ni atom. The high activity and fast reactivation, together with a degree of oxygen tolerance for the H-2-production activity, make the Hys hydrogenases attractive catalysts for technological applications.