In symbiosis with peas, Rhizobium leguminosarum UPM791 induces the synthesis of a hydrogen uptake (Hup) system that recycles hydrogen generated in nodules by nitrogenase. A cosmid (pAL618) containing hup genes from this strain on a 20-kilobase-pair (kb) DNA insert has previously been isolated in our laboratory (A. Leyva, J. M. Palacios, T. Mozo, and T.Ruiz-Argüeso, J. Bacteriol. 169:4929-4934, 1987). Here we show that cosmid pAL618 contains all of the genetic information required to confer high levels of hydrogenase activity on the naturally Hup- strains R. leguminosarum UML2 and Rhizobium phaseoli CFN42, and we also describe in detail the organization of hup genes on pAL618. To study hup gene organization, site-directed transposon mutagenesis and complementation analysis were carried out. According to the Hup phenotype associated with the transposon insertions, hup genes were found to span a 15-kilobase-pair region within pAL618 insert DNA. Complementation analysis revealed that Hup- mutants fell into six distinct complementation groups that define six transcriptional units, designated regions hupI to hupVI. Region hupI was subcloned and expressed in Escherichia coli cells under the control of a bacteriophage T7 promoter. A polypeptide of ca. 65 kilodaltons that was cross-reactive with antiserum against the large subunit of Bradyrhizobium japonicum hydrogenase was detected both in E. coli cells carrying the cloned hupI region and in pea bacteroids from strain UPM791, indicating that region hupI codes for structural genes of R. leguminosarum hydrogenase.