The hypCD genes, encoding the counterparts of mesophilic proteins involved in the maturation of [NiFe] hydrogenases, were isolated from the hyperthermophilic archaeon Thermococcus litoralis. The deduced gene products showed 30-40% identity to the corresponding mesophilic proteins. HypC and HypD were synthesized by the T7 expression system. Heterologous complementation experiments were done in Escherichia coli and Ralstonia eutropha strains lacking functionally active hypC and hypD genes. Only the cytoplasmic hydrogenase of R. eutropha could be processed by HypD from T. litoralis. This was the first demonstration of mesophilic hydrogenase processing using a hyperthermophilic archaeal accessory protein to produce an active enzyme.