The TOL plasmid pWW53 encodes a catabolic pathway for the metabolism of toluene. It bears an upper-pathway operon for the oxidation of toluene to benzoate and a copy of the gene that encodes regulatory protein XylR. For metabolism of the aromatic carboxylic acid, it bears two functional homologous meta-pathway operons, together with two functional copies of the xylS regulatory gene (xylS1 and xylS3). In cells growing in the absence of pathway substrates, no mRNA from upper- and meta-pathway operons were found; however, the xylR gene was expressed from two sigma70-dependent tandem promoters, and the xylS1 and the xylS3 genes were also expressed from their sigma70-dependent promoters, called Ps2 and Ps3, respectively. In cells grown in the presence of o-xylene, the XylR protein became active and stimulated transcription from the Pu promoter for the upper pathway. Expression from xylS1 but not from xylS3 was also stimulated by XylR; this was due to activation of transcription from the xylS1 Ps1 promoter, which is sigma54 dependent, and the lack of effect on expression from the Ps2 sigma70-dependent promoter. As a result of overexpression of the xylS1 gene, the XylS1 protein was overproduced and activated transcription from Pm1 and Pm2. In cells growing on benzoate, the upper-pathway operon was not expressed, but both meta operons were expressed. Given that XylS1 but not XylS3 recognized benzoate as an effector, stimulation of transcription was found to be mediated by XylS1. This was confirmed with cloned meta-pathway promoters and regulators. When 3-methylbenzoate was present in the medium, both meta operons were also expressed and stimulation of transcription was mediated by both XylS1 and XylS3, which both recognized 3-methylbenzoate as an effector.