A 25 kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced. Coding regions corresponding to 20 open reading frames were identified. The R. capsulatus hydrogenase-specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium leguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11). The sequential arrangement of the R. capsulatus genes is: hupR2-hupU-hypF-hupS-hupL-hupM-hu pD-hupF-hupG-hupH-hupJ-hupK-hypA- hypB-hupR1- hypC-hypD-hypE-ORF19-ORF20, all contiguous and transcribed from the same DNA strand. The last two potential genes do not encode products that are related to identified hydrogenase-specific gene products in other species. The sequence of the 12 R. capsulatus genes underlined above is presented. The mutation site in two of the Hup- mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD gene (deletion of 54 bp), respectively. The hypF gene product shares 45% identity with the product of hydA from E. coli and the product of hypF from R. leguminosarum. Those products present at their N-terminus a Cys arrangement typical of zinc-finger proteins. The G deletion in the C-terminal region of hypF in the RS13 mutant prevented the expression of a hupS::lacZ translational fusion from being stimulated by H2 as it is observed in the wild-type strain B10. It is inferred that the HypF protein is a factor involved in H2 stimulation of hydrogenase expression.