Hydrogenase from C. pasteurianum is an iron-sulfur protein containing at least two tetrameric iron-sulfur centers. Information on the structure of the remaining iron atoms must await future investigation. Although the EPR spectra of dithionite-reduced hydrogenase and eight-iron Fd showed some similarity, the CD spectra clearly indicated a difference. The tetrameric iron-sulfur centers of hydrogenase were shown to undergo redox changes when hydrogenase was oxidized or reduced. However, no evidence is now available to support a role for the tetrameric Fe-S centers, responsible for the EPR spectrum A, as the primary site for H2 binding and activation. Because we have found that the [Fe4S4(SR)4]-containing ferredoxins do not have hydrogenase activity, it is conceivable that the additional iron atoms and/or certain amino acid residues of hydrogenase also contribute to the unique catalytic properties of this enzyme. Chemical synthesis of Fe-S clusters with different peptide environments and with hydrogenase function would lead to the identification of these functional groups. X-ray diffraction studies on hydrogenase will certainly complement the other approaches. Knowledge of the structure of the active site of hydrogenase will certainly accelerate research into: (1) the synthesis of a stable catalyst to replace hydrogenase in systems designed to produce H2 by coupling this catalyst to a photoreducing system; and (2) the elucidation of the active sites of more complicated iron-sulfur enzymes such as nitrogenase.