The regulation of the tou operon of Pseudomonas stutzeri OX1, for degradation of toluene and o-xylene via phenolic intermediates, has been faithfully reconstructed in vitro with purified proteins. The set-up included the prokaryotic enhancer-binding protein TouR, the sigma54-dependent PToMO promoter and the sigma54-containing RNA polymerase. With this system we prove that direct binding of 2-methylphenol (o-cresol) to TouR is the only regulatory step for activation of PToMO in response to aromatic effectors, thereby ruling out the involvement of other factors or a need for protein processing. In addition, we found that while TouR failed entirely to activate PToMO in the absence of inducers, the protein had per se a very significant ATPase activity, which was only moderately increased by o-cresol addition. The results presented here support the view that TouR-like proteins are particularly suitable as evolutionary assets to endow recently evolved pathways for the degradation of environmental pollutants with an optimal degree of transcriptional regulation.