A polyphasic study was carried out to determine the taxonomic position of two aerobic, cyanide-degrading bacterial strains, designated L61T and L22, which had been isolated from a bioreactor for the treatment of nickel-complexed cyanide. The two isolates exhibited almost identical taxonomic characteristics. Phylogenetic analysis inferred from comparative 16S rRNA gene sequences indicated that the isolates fall in a sublineage of the genus Rhizobium comprising the type strains of Rhizobium giardinii, Rhizobium radiobacter, Rhizobium rubi, Rhizobium larrymoorei, Rhizobium vitis, Rhizobium undicola, Rhizobium loessense, Rhizobium galegae and Rhizobium huautlense. Cells of the two isolates are Gram-negative, aerobic, motile and non-spore-forming rods (0.6-0.7x1.1-1.3 microm), with peritrichous flagella. The DNA G+C content is 60.1-60.9 mol%. Cellular fatty acids are C(16 : 0) (2.2-3.3 %), C(18 : 0) (2.1-3.2 %), C(19 : 0) cyclo omega8c (9.9-16.8 %), C(20 : 3)omega6,9,12c (2.7-3.3 %), summed feature 3 (7.2-7.7 %) and summed feature 7 (67.8-73.7 %). The strains formed nodules on a legume plant, Medicago sativa. A nifH gene encoding denitrogenase reductase, the key component of the nitrogenase enzyme complex, was detected in L61T by PCR amplification by using a nifH-specific primer system. Strains L61T and L22 were distinguished from the type strains of recognized Rhizobium species in the same sublineage based on low DNA-DNA hybridization values (2-4 %) and/or a 16S rRNA gene sequence similarity value of less than 96 %. Moreover, some phenotypic properties with respect to substrate utilization as a carbon or nitrogen source, antibiotic resistance and growth conditions could be used to discriminate L61T and L22 from Rhizobium species in the same sublineage. Based on the results obtained in this study, L61T and L22 are considered to be representatives of a novel species of Rhizobium, for which the name Rhizobium daejeonense sp. nov. is proposed. The type strain is L61T (=KCTC 12121T=IAM 15042T=CCBAU 10050T).