The mechanism by which XylR, the toluene-responsive activator of the σ54-dependent Pu and Ps promoters of the Pseudomonas TOL plasmid pWW0, downregulates its own σ70 promoter Pr has been examined. An in vitro transcription system was developed in order to reproduce the repression of Pr observed in cells of P. putida (pWW0) both in the presence and in the absence of the XylR inducer, benzyl alcohol. DNA templates bearing the two σ70-RNA polymerase (RNAP) binding sites of Pr, which overlap the upstream activating sequences (UAS) for XylR in the divergent σ54 promoter Ps, were transcribed in the presence of a constitutively active XylR variant deleted of its N-terminal domain (XylRΔA). The addition of ATP, known to trigger multimerization of the regulator at the UAS, enhanced the repression of Pr by XylR. Furthermore, we observed activation of the divergent σ54 promoter Ps during Pr downregulation by XylRΔA. These results support the notion that activation of XylR by aromatic inducers in vivo triggers a transcriptional switch between Pr and PsSuch a switch is apparently caused by the ATP-dependent multimerization and strong DNA binding of the protein required for activation of the σ54 promoter. This device could reset the level of XylR expression during activation of the σ54Pu and Ps promoters of the TOL plasmid.