The plasmids pDN211 and pDNll, isolated from the gene bank of the Rhizobium japonicum strain I-110, have been reported to complement two different Nif+ Hup· (nitrogen fixation positive and hydrogen uptake negative) mutants. A 5.9-kb Hindiii DNA fragment of the cosmid pHU52, isolated from the gene bank of R. japonicum strain 122DES, has been reported to code for the two polypeptide subunits of uptake hydrogenase. To determine homology between the structural genes of uptake hydrogenase of the two strains, a Southern blot of the Hindiii restriction fragments of the plasmids pDN211 and pDN11 was hybridized to the 5.9-kb Hindiii fragment. A 6.0-kb HindIII DNA fragment of pDN11 was observed to be homologous to the hup DNA probe. Thus, the hup genes of the two Rhizobium strains are conserved. Colony hybridization with the 5.9-kb DNA as the probe was used to detect the homologous hup genes in alfalfa-, chickpea- and pigeonpea- Rhizobium species. These Rhizobium species were also successfully derepressed for uptake hydrogenase in free living conditions. It was found that 30% of the alfalfa-, 30% of the chickpea- and 21% of the pigeonpea- Rhizobium strains tested were Hup+ as determined by the methylene blue (MB) reduction assay. All but one strain of alfalfa- (Celpril Ind. 3623) and one strain of pigeonpea- Rhizobium (IC3282) that showed strong homology to the hup DNA probe also exhibited MB reduction activity. The Hup+ strains of alfalfa- and pigeonpea- Rhizobium produced significantly higher yields as compared to the Hup- strains, whereas those of the chickpea-Rhizobium strains produced significantly lower yields as compared to the Hup- strains. Two of the alfalfa-Rhizobium strains, USDA1024 and CmRm~, exhibited Hup activities greater than any reported previously for this bacterial species. The cosmid-borne hup genes of R. japonicum were successfully expressed in all strains tested but the enzyme activities were very low in alfalfa-Rhizobium compared to those in chickpea- and pigeonpea-Rhizobium species. The relative efficiency of N2-fixation was significantly increased by the transfer of hup genes into the chickpea- and pigeonpea- Rhizobium strains.