The 58/59 min region of the Escherichia coli chromosome contains two divergently oriented gene clusters coding for proteins with a function in hydrogenase formation. One cluster (the hyc operon), transcribed counterclockwise with respect to the E. coli chromosome, codes for gene products with a structural role in hydrogenase 3 formation (Böhm et al., 1990). The nucleotide sequence of the divergently transcribed operon (hyp) has been determined. It contains five genes, all of which are expressed in vivo in a T7 promoter/polymerase system, and the sizes of the synthesized products correspond with those predicted from the amino acid sequence. Complementation analysis of previously characterized mutants showed that the hypB, hypC and hypD genes have a function in the formation of all three hydrogenase isoenzymes, lesions in hypB being complemented by high nickel ion concentration in the medium. Prevention of hypBCDE gene expression led to an altered electrophoretic pattern of hydrogenase 1 and 2 constituent subunits, indicating increased chemical or proteolytic subunits, Under fermentative growth conditions, operon expression was governed by an NtrA-dependent promoter lying upstream of hypA working together with an fnr gene product-dependent promoter which was localized within the hypA gene. The latter (operon-internal) promoter is responsible for hypBCDE transcription under non-fermentative conditions when the -24/-12 NtrA-dependent promoter upstream of hypA is silent.