Plasmid pP51 of Pseudomonas sp. strain P51 contains two gene clusters encoding the degradation of chlorinated benzenes, tcbAB and tcbCDEF. A regulatory gene, tcbR, was located upstream and divergently transcribed from the chlorocatechol oxidative gene cluster tcbCDEF. The tcbR gene was characterized by DNA sequencing and expression studies with Escherichia coli and pET8c and appeared to encode a 32-kDa protein. The activity of the tcbR gene product was analyzed in Pseudomonas putida KT2442, in which it appeared to function as a positive regulator of tcbC expression. Protein extracts of both E. coli overproducing TcbR and Pseudomonas sp. strain P51 showed specific DNA binding to the 150-bp region that is located between the tcbR and tcbC genes. Primer extension mapping demonstrated that the transcription start sites of tcbR and tcbC are located in this region and that the divergent promoter sequences of both genes overlap. Amino acid sequence comparisons indicated that TcbR is a member of the LysR family of transcriptional activator proteins and shares a high degree of homology with other activator proteins involved in regulating the metabolism of aromatic compounds.