The pathway of anaerobic degradation ofo-phthalate was studied in the nitrate-reducing bacteriumAzoarcussp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins ino-phthalate-grown compared to benzoate-grown cells. The genes encodingo-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome ofAzoarcussp. strain PA01. Theo-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA-transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobico-phthalate degradation. We propose thato-phthalate is first activated too-phthalyl-CoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, ando-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putativeo-phthalyl-CoA decarboxylase. Results fromin vitroenzyme assays with cell-free extracts ofo-phthalate-grown cells demonstrated the formation ofo-phthalyl-CoA fromo-phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl-CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity foro-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o-phthalate whereas the putativeo-phthalyl-CoA decarboxylase converted fluoro-o-phthalyl-CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygen-insensitive and inducible only after growth witho-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria.