Mutants of Pseudomonas putida mt-2 that are unable to convert benzoate to catechol were isolated and grouped into two classes: those that did not initiate attack on benzoate and those that accumulated 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (benzoate diol). The latter mutants, represents by strain PP0201, were shown to lack benzoate diol dehydrogenase (benD) activity. Mutants from the former class were presumed either to carry lesions in one or more subunit structural genes of benzoate dioxygenase (benABC) or the regulatory gene (benR) or to contain multiple mutations. Previous work in this laboratory suggested that benR can substitute for the TOL plasmid-encoded xylS regulatory gene, which promotes gene expression from the OP2 region of the lower or meta pathway operon. Accordingly, structural and regulatory gene mutations were distinguished by the ability of benzoate-grown mutant strains to induce expression from OP2 without xylS by using the TOL plasmid xylE gene (encoding catechol 2,3-dioxygenase) as a reporter. A cloned 12-kb BamHI chromosomal DNA fragment from the P. aeruginosa PAO1 chromosome complemented all of the mutations, as shown by restoration of growth on benzoate minimal medium. Subcloning and deletion analyses allowed identification of DNA fragments carrying benD, benABC, and the region possessing xylS substitution activity, benR. Expression of these genes was examined in a strain devoid of benzoate-utilizing ability, Pseudomonas fluorescens PFO15. The disappearance of benzoate and the production of catechol were determined by chromatographic analysis of supernatants from cultures grown with casamino acids. When P. fluorescens PFO15 was transformed with plasmids containing only benABCD, no loss of benzoate was observed. When either benR or xylS was cloned into plasmids compatible with those plasmids containing only the benABCD regions, benzoate was removed from the medium and catechol was produced. Regulation of expression of the chromosomal structural genes by benR and xylS was quantified by benzoate diol dehydrogenase enzyme assays. The results obtained when xylS was substituted for benR strongly suggest an isofunctional regulatory mechanism between the TOL plasmid lower-pathway genes (via the OP2 promoter) and chromosomal benABC. Southern hybridizations demonstrated that DNA encoding the benzoate dioxygenase structural genes showed homology to DNA encoding toluate dioxygenase from the TOL plasmid pWW0, but benR did not show homology to xylS. Evolutionary relationships between the regulatory systems of chromosomal and plasmid-encoded genes for the catabolism of benzoate and related compounds are suggested.