The hydA locus of Escherichia coli is known to encode some function necessary for formation of hydrogenase activity. The locus contains two open reading frames, hydN and hypF. In this communication, an analysis of the regulation of these two genes and of the phenotype of respective mutants is presented, Both genes were expressed in a T7 promoter/polymerase system, yielding a 19-kDa (HydN) and a 81-kDa (HypF) protein. In-frame deletions were constructed for each gene and transferred to the chromosome by homologous recombination. The mutation in hydN led to a decrease of the activity of formate dehydrogenase H (FDH-H) in crude extracts, but the activity and maturation of hydrogenases were nearly unaffected. In contrast, a deletion in hypF resulted in the loss of hydrogenase activity and in the synthesis of the large subunits of the hydrogenase isoenzymes 1, 2 and 3 in the inactive precursor form. For hydrogenase 3, it was shown that this is due to a lack of incorporation of nickel into the large subunit. hydN and hypF are organised in an operon that is a member of the formate regulon. Transcription was shown to be dependent on sigma 54 and FhlA, and an FhlA-binding site upstream of hydN was identified. The sigma 54-dependent promoter shows a rare deviation from the consensus at positions -24/-12, namely GG/GA instead of GG/GC. In conclusion, the product of hydN appears to have some role in electron flow from or to FDH-H, and the product of hypF is connected with maturation of all three hydrogenases of E. coli.