Two 2,3-dihydroxybiphenyl (23DHBP) dioxygenase genes, bphC1 and etbC involved in the degradation of polychlorinated biphenyl(s) (PCBs) have been isolated and characterized from a strong PCB degrader, Rhodococcus sp. RHA1. In this study, four new 23DHBP dioxygenase genes, designated as bphC2, bphC3, bphC4, and bphC5 were isolated from RHA1, and their nucleotide sequences were determined. Based on amino acid sequence similarities, all of the newly isolated bphC genes could be categorized into type I along with BphC1 and EtbC [Eltis, L.D. and Bolin, J.T., J. Bacteriol., 178, 5930-5937 (1996)]. Six bphC genes, including bphC1, etbC, and four new genes, were expressed in Escherichia coli to determine their substrate specificity. The activities of BphC2, BphC3, BphC4, and BphC5 were found to be specific to 23DHBP, while BphC1 and EtbC exhibited activities towards compounds other than 23DHBP, including catechol (CAT) and 3-methylcatechol (3MC). RNA slot blot hybridization analysis indicated that only bphC5 was transcribed among the newly isolated bphC in RHA1 cells grown on biphenyl and ethylbenzene. The nucleotide sequence of the flanking region of each bphC revealed a homolog of the 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase gene, bphD, just upstream of bphC5. The bphC5 and putative bphD genes may constitute an operon and play a role in the degradation of biphenyl and PCBs together with bphC1 and etbC. In contrast, the bphC2, bphC3, and bphC4 genes may not be involved in biphenyl and PCB degradation.