Hydrogen-uptake (Hup) activity in Azotobacter chroococcum depends upon a cluster of genes spread over 13,687 bp of the chromosome. Six accessory genes of the cluster, hupABYCDE, begin 4.8 kb downstream of the structural genes, hupSL, and are required for the formation of a functional [NiFe] hydrogenase. The sequencing of the intervening 4.8 kb of hup-specific DNA has now been completed. This revealed eight additional closely linked ORFs, which we designated hupZ, hupM, hupN, hupO, hupQ, hupR, hupT and hupV. These genes potentially encode polypeptides with predicted masses of 27.7, 22.3, 11.4, 16.2, 31.3, 8.1, 16.2 and 36.7 kDa, respectively. All eight genes are transcribed from the same strand as hupSL and hupABYCDE. A chroococcum, therefore, has a total of 16 contiguous genes affecting hydrogenase activity beginning with hupS and ending with hupE. The amino acid sequence deduced from hupZ has the characteristics of a b-type cytochrome. Insertion mutagenesis of hupZ resulted in a mutant incapable of supporting O2-dependent H2 oxidation. The deduced amino acid sequence of hupR shares high homology with bacterial rubredoxins. HupZ and HupR may both be involved in transferring electrons from hydrogenase to the electron transport chain. A mutation in hupV knocked out hydrogenase activity entirely; this gene may be involved in processing the large subunit of hydrogenase. It is now clear that the genes controlling [NiFe] hydrogenase activity in many bacteria including Azotobacter chroococcum, Alcaligenes eutrophus, Rhizobium leguminosarum, Rhodobacter capsulatus and Escherichia coli are highly conserved, organized in much the same manner, and likely derived from a common ancestor.