The regulatory hydrogenase (RH) from Ralstonia eutropha acts as the H-2-sensing unit of a two-component system that regulates biosynthesis of the energy conserving hydrogenases of the organism according to the availability of H-2. The H-2 oxidation activity, which was so far determined in vitro with artificial electron acceptors, has been considered to be insensitive to O-2 and CO. It is assumed that bulky isoleucine and phenylalanine amino acid residues close to the NiFe active site "gate" gas access, preventing molecules larger than H-2 interacting with the active site. We have carried out sensitive electrochemical measurements to demonstrate that O-2 is in fact an inhibitor of H-2 oxidation by the RH, and that both H+ reduction and H-2 oxidation are inhibited by CO. Furthermore, we have demonstrated that the inhibitory effect of O-2 arises due to interaction of O-2 with the active site. Using protein film infrared electrochemistry (PFIRE) under H-2 oxidation conditions, in conjunction with solution infrared measurements, we have identified previously unreported oxidized inactive and catalytically active reduced states of the RH active site. These findings suggest that the RH has a rich active site chemistry similar to that of other NiFe hydrogenases.