The hupM gene, previously called ORFX, found downstream from and contiguous with the structural hydrogenase genes hupS and hupL in Rhodobacter capsulatus, is shown here to form a single hupSLM transcription unit with the two other genes. The hupM gene was inactivated by interposon mutagenesis. The two selected mutants, BCX1 and BCX2, which contained the kanamycin-resistance gene in opposite orientation, still exhibited hydrogenase activity when assayed with the artificial electron acceptors benzylviologen and methylene blue. However, the hydrogenase was not physiologically active in these mutants, which could not grow autotrophically and were unable to recycle electrons to nitrogenase or to respire on H2. The hupM gene starts nine base pairs downstream from the TGA stop codon of hupL gene, which encodes the large subunit of the [NiFe]hydrogenase of Rhodobacter capsulatus. The three contiguous genes hupS, hupL and hupM were subcloned downstream from the promoter of hupSL, either with the promoter in the correct orientation (plasmid pBC8) or with the promoter in the opposite orientation (plasmid pBC9), then the constructs were introduced into the mutant strains. Only plasmid pBC8 could restore the formation of a competent hydrogenase in mutants BCX1 and BCX2, indicating that the hupM gene is expressed only from the hupSL promoter.