We isolated Comamonas sp. strain E6, which utilizes terephthalate (TPA) as the sole carbon and energy source via the protocatechuate (PCA) 4,5-cleavage pathway. Two almost identical TPA degradation gene clusters, tphRICIA2IA3IBIA1I and tphRIICIIA2IIA3IIBIIA1II, were isolated from this strain. Based on amino acid sequence similarity, the genes tphR, tphC, tphA2, tphA3, tphB, and tphA1 were predicted to code, respectively, for an IclR-type transcriptional regulator, a periplasmic TPA binding receptor, the large subunit of the oxygenase component of TPA 1,2-dioxygenase (TPADO), the small subunit of the oxygenase component of TPADO, a 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD) dehydrogenase, and a reductase component of TPADO. The growth of E6 on TPA was not affected by disruption of either tphA2I or tphA2II singly; however, the tphA2I tphA2II double mutant no longer grew on TPA, suggesting that both TPADO genes are involved in TPA degradation. Introduction of a plasmid carrying tphRIICIIA2IIA3IIBIIA1II conferred the TPA utilization phenotype on Comamonas testosteroni IAM 1152, which is able to grow on PCA but not on TPA. Disruption of either tphRII or tphCII on this plasmid resulted in the loss of the growth of IAM 1152 on TPA, suggesting that these genes are essential to convert TPA to PCA in E6. The genes tphA1II, tphA2II, tphA3II, and tphBII were expressed in Escherichia coli, and the resultant cell extracts containing TphA1II, TphA2II, and TphA3II converted TPA in the presence of NADPH into a product which was transformed to PCA by TphBII. On the basis of these results, TPADO was strongly suggested to be a two-component dioxygenase which consists of the terminal oxygenase component (TphA2 and TphA3) and the reductase (TphA1), and tphB codes for the DCD dehydrogenase.