Iron sulphur clusters (FeS) are essential cofactors in the small, electron-transfer subunits of [NiFe]-hydrogenases (Hyd). In this study we analyzed the in vivo role of ferredoxin in the biosynthesis of three of the Hyd in Escherichia coli. Our results reveal that a fdx mutant, which is unable to synthesize ferredoxin, lacks the activity of both hydrogen-oxidizing enzymes Hyd-1 and Hyd-2. In the case of Hyd-2 this was due to the absence of the FeS cluster-containing small subunit. In the case of Hyd-1, stability of the catalytic subunit was also impaired. Partial activity of the hydrogen-evolving Hyd-3 enzyme, as well as that of both respiratory formate dehydrogenases was retained in the fdx mutant. Analysis of lacZ fusions demonstrated that the fdx mutation had a limited effect on expression of the operon encoding Hyd-1. Rather, these data suggest that ferredoxin has a role in the maturation or assembly of the hydrogen-oxidizing [NiFe]-hydrogenases. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.