Pseudomonas sp. strain PTH10 can utilize o-phthalate which is a key intermediate in the bacterial degradation of some polycyclic aromatic hydrocarbons. In this strain, o-phthalate is degraded to 2,3-dihydroxybenzoate and further metabolized via the 2,3-dihydroxybenzoate meta-cleavage pathway. Here, the opa genes which are involved in the o-phthalate catabolism were identified. Based on the enzymatic activity of the opa gene products, opaAaAbAcAd, opaB, opaC, and opaD were found to code for o-phthalate 2,3-dioxygenase, dihydrodiol dehydrogenase, 2,3-dihydroxybenzoate 3,4-dioxygenase, and 3-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase, respectively. Collectively, these enzymes are thought to catalyze the conversion of o-phthalate to 2-hydroxymuconate-6-semialdehyde. Deletion mutants of the above opa genes indicated that their products were required for the utilization of o-phthalate. Transcriptional analysis showed that the opa genes were organized in the same transcriptional unit. Quantitative analysis of opaAa, opaB, opaC, opaD, opaE, and opaN revealed that, except for opaB and opaC, all other genes were transcriptionally induced during growth on o-phthalate. The constitutive expression of opaB and opaC, and the transcriptional induction of opaD located downstream of opaB, suggest several possible internal promoters are existence in the opa cluster. Together, these results strongly suggest that the opa genes are involved in a novel o-phthalate catabolic pathway in strain PTH10.