The regulation of the DNT pathway for biodegradation of 2,4-dinitrotoluene of Burkholderia sp. DNT has been examined by exporting each of its components to Pseudomonas putida KT2440. The cognate regulator DntR does not respond to the pathway substrate, but to the non-substrate salicylate. In order to examine whether such a response to an unrelated inducer was specific or rather a vestige of a previous evolutionary stage, the complete dnt complement or parts of it were expressed functionally for accumulation of various metabolic intermediates. Their effect on expression of dnt genes was then followed both biochemically and by means of a luminescent reporter engineered in the surrogate host. DntR was not only unresponsive to DNT biodegradation products, but it also failed to influence expression of dnt genes at all. Comparison of the dntR/dntA divergent promoter region with similar ones found in various catabolic systems indicated that the leading segment of the DNT biodegradation pathway evolved from a matching portion of naphthalene biodegradation routes existing in other bacteria. That a useless but still active transcriptional factor occurs along enzymes that have already evolved a new substrate specificity suggests that emergence of novel catalytic abilities precedes their submission to cognate regulatory devices, not vice versa.