The archaeon Methanococcus voltae encodes two pairs of NiFe-hydrogenase isoenzymes. One hydrogenase of each pair contains selenium in the active site, whereas the other one is selenium-free. The gene groups for the selenium-free hydrogenases, called vhc and frc, are linked by a common intergenic region. They are only transcribed under selenium limitation. A protein binding to a negative regulatory element involved in the regulation of the two operons was purified by DNA-affinity chromatography. Through the identification of the corresponding gene the protein was found to be a LysR-type regulator. It was named HrsM (hydrogenase gene regulator, selenium dependent in M. voltae). hrsM knockout mutants constitutively transcribed the vhc and frc operons in the presence of selenium. A putative HrsM binding site was also detected in the intergenic region in front of the hrsM gene. Northern blot analysis indicated that the hrsM gene might be autoregulated.