AbstractAn R‐prime plasmid (pJB3JI‐18) carrying a 10 kb region of Azorhizobium caulinodans ORS571 DNA which codes for uptake hydrogenase activity was isolated from a Km8 derivative of R68.45. R‐prime plasmids were obtained by transferring R68.45 to ORS571, retransferring it to E. coli, and selecting transconjugants for hydrogenase activity by means of a colour test based on methylene blue. The R‐prime plasmid DNA was hybridized with a DNA region which contained the hup genes of Bradyrhiobium japonicum, indicating homology between the heterologous hup genes. Transfer of the R‐prime plasmid to different Hup− mutants restored the Hup+ phenotype. When it was introduced to R. meliloti strains, it maintained its molecular weight and did not affect indigenous plasmids in two of three recipients. In the third recipient cointegrate formation and incompatibility with indigenous plasmids were found.