[NiFe]-hydrogenases (Hyd) bind a nickel-iron-based cofactor. The Fe ion of the cofactor is bound by two cyanide ligands and a single carbon monoxide ligand. Minimally six accessory proteins (HypA-HypF) are necessary for NiFe(CN)2CO cofactor biosynthesis in Escherichia coli. It has been shown that the anaerobically purified HypC-HypD-HypE scaffold complex carries the Fe(CN)2CO moiety of this cofactor. In the present study, we have purified the HybG-HypDE complex and used it to successfully reconstitute in vitro active Hyd from E. coli. HybG is a homologue of HypC that is specifically required for the maturation of Hyd-2 and also functions in the maturation of Hyd-1 of E. coli. Maturation of active Hyd-1 and Hyd-2 could be demonstrated in extracts derived from HybG- and HypD-deficient E. coli strains by adding anaerobically purified HybG-HypDE complex. In vitro maturation was dependent on ATP, carbamoylphosphate, nickel and reducing conditions. Hydrogenase maturation was prevented when the purified HybG-HypDE complex used in the maturation assay lacked a bound Fe(CN)2CO moiety. These findings demonstrate that it is possible to isolate incompletely processed intermediates on the maturation pathway and to use these to activate apo-forms of [NiFe]-hydrogenase large subunits.