The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H2 at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous, but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-VIS absorption spectrum as well as by shifts of the Fe K-edge from X-ray absorption spectroscopy (XAS) in the wild-type double dimeric RHWT [HoxBC]2 and in a monodimeric derivative designated RHstop, lacking the C-terminal 55 amino acids of HoxB. According to analysis of Fe EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RHWT seems to harbour two [2Fe-2S] clusters in addition to a 4Fe-species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RHstop. Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain which involves complex formation between [HoxBC]2 and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.