Both 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) and 2-hydroxy-6-oxohepta-2,4-dienoic acid (HOHD) hydrolases were purified to homogeneity from the cells of PCB-degrader, Rhodococcus sp. RHA1 grown on biphenyl. The NH2-terminal amino acid sequences of these hydrolases were determined and low homology (35%) was observed between them. Both of them were induced in the RHA1 cells grown on benzene, toluene and ethylbenzene as well as on biphenyl, but not induced in those grown on benzoate, phenol and succinate. The RHA1 HPDA and HOHD hydrolases were indicated to be an octamer and a dimer respectively composed of identical subunits with a molecular weight of 33,000 and 32,000, respectively. The RHA1 HPDA and HOHD hydrolases were specific for the respective substrates HPDA and HOHD, and the latter also had high activity toward 2-hydroxy muconic semialdehyde (HMSA). Such narrow substrate specificity suggested that HPDA and HOHD hydrolases were responsible for the degradation of biphenyl and toluene, respectively.