Three groups that absorb in the 2100–1800‐cm−1 infrared spectral region have recently been detected in Ni‐hydrogenase from Chromatium vinosum [Bagley, K. A., Duin, E. C., Roseboom, W., Albracht, S. P. J. & Woodruff, W. H. (1995) Biochemistry 34, 5527–5535]. To assess the significance and generality of this observation, we have carried out an infrared‐spectroscopic study of eight hydrogenases of three different types (nickel, iron and metal‐free) and of 11 other iron‐sulfur and/or nickel proteins. Infrared bands in the 2100–1800‐cm−1 spectral region were found in spectra of all Ni‐hydrogenases and Fe‐hydrogenases and were absent from spectra of any of the other proteins, including a metal‐free hydrogenase. The positions of these bands are dependent on the redox state of the hydrogenase. The three groups in Ni‐hydrogenases that are detected by infrared spectroscopy are assigned to the three unidentified small non‐protein ligands that coordinate iron in the dinuclear Ni/Fe active site as observed in the X‐ray structure of the enzyme from Desulfovibrio gigas [Volbeda, A., Charon, M.‐H., Piras, C., Hatchikian, E. C., Frey, M. & Fontecilla‐Camps, J. C. (1995) Nature 373, 580–587]. It is concluded that these groups occur exclusively in metal‐containing H2‐activating enzymes. It is proposed that the active sites of Ni‐hydrogenases and of Fe‐hydrogenases have a similar architecture, that is required for the activation of molecular hydrogen.