The study of Ni-Fe-Se hydrogenases is interesting from the basic research point of view because their active site is a clear example of how nature regulates the catalytic function of an enzyme by the change of a single residue, in this case a cysteine, which is replaced by a selenocysteine. Most hydrogenases are inhibited by CO and O(2). In this work we studied these inhibition processes for the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough by combining catalytic activity measurements, followed by mass spectrometry or chronoamperometry, with Fourier transform IR spectroscopy experiments. The results show that the CO inhibitor binds to Ni in both conformations of the active site of this hydrogenase in a way similar to that in standard Ni-Fe hydrogenases, although in one of the CO-inhibited conformations the active site of the Ni-Fe-Se hydrogenase is more protected against the attack by O(2). The inhibition of the Ni-Fe-Se hydrogenase activity by O(2) could be explained by oxidation of the terminal cysteine ligand of the active-site Ni, instead of the direct attack of O(2) on the bridging site between Ni and Fe.