Rhizobium huakuii bv. renge, which is able to produce nodules on Astragalus sinicus cv. Japan, has been assigned to a new biovariety based on its host range and taxonomic characteristics. We reproducibly demonstrated the presence of an indigenous large plasmid, with a molecular mass of about 260 Mda, in R. huakuii bv. renge and named the plasmid pRhYM. Using a heat-curing procedure, we obtained derivative strains of R. huakuii bv. renge that were defective for nodulation (Nod(-)) and nitrogen fixation (Fix(-)). Nod(-) mutant strains either did not contain pRhYM or had deletions of pRhYM segments. [P-32]-Labeled probes of cloned DNA fragments of nod and nif from Rhizobium sp. NGR234 and Klebsiella pneumoniae, respectively, hybridized to pRhYM of R. huakuii bv. renge, but not to DNA from most of the Nod(-) mutant strains. These results show that the nod and nif genes of R. huakuii bv. renge are located on pRhYM.