Evolutionary expansion of metabolic networks entails the emergence of regulatory factors that become sensitive to new chemical species. A dedicated genetic system was developed for the soil bacterium Pseudomonas putida aimed at deciphering the steps involved in the gain of responsiveness of the toluene-activated prokaryotic regulator XylR to the xenobiotic chemical 2,4 dinitrotoluene (DNT). A mutant library of the A domain of XylR was screened in vivo for those variants activated by DNT through coupling the cognate promoter Pu to the P. putida yeast URA3 homologue, pyrF. All DNT-responsive clones maintained their sensitivity to ordinary effectors of XylR and broadened the range of inducers to unrelated aromatics. Yet, none of the altered amino acids lay in the recognizable effector binding pocket of the polypeptide. Instead, mutations appeared in protein surfaces believed to engage in the conformational shifts that follow effector binding and modulate signal transmission between XylR domains. It thus seems that transcriptional factors are likely to regress into functionally multipotent forms (i.e. stem protein types) as a first step towards the divergence of a new specificity.