Many toxic man-made compounds have been introduced into the environment, and bacterial strains that are able to grow on them are ideal model systems for studying the evolution of metabolic pathways and regulatory systems. Acidovorax sp. strain JS42 is unique in its ability to use 2-nitrotoluene as a sole carbon, nitrogen, and energy source for growth. The LysR-type transcriptional regulator NtdR activates expression of the 2-nitrotoluene degradation genes not only when nitroaromatic compounds are present, but also in the presence of a wide range of aromatic acids and analogues. The molecular determinants of inducer specificity were identified through comparative analysis with NagR, the activator of the naphthalene degradation pathway genes in Ralstonia sp. strain U2. Although NagR is 98% identical to NtdR, it does not respond to nitrotoluenes. Exchange of residues that differ between NagR and NtdR revealed that residues at positions 227 and 232 were key for the recognition of nitroaromatic compounds, while the amino acid at position 169 determined the range of aromatic acids recognized. Structural modelling of NtdR suggests that these residues are near the predicted inducer binding pocket. Based on these results, an evolutionary model is presented that depicts the stepwise evolution of NtdR.