Acidovorax (formerly Pseudomonas) sp. strain JS42 utilizes 2-nitrotoluene as sole carbon, nitrogen, and energy source. 2-Nitrotoluene 2,3-dioxygenase (2NTDO) catalyzes the initial step in 2-nitrotoluene degradation by converting 2-nitrotoluene to 3-methylcatechol. In this study, we identified specific amino acids at the active site that control specificity. The residue at position 350 was found to be critical in determining both the enantiospecificity of 2NTDO with naphthalene and the ability to oxidize the ring of mononitrotoluenes. Substitution of Ile350 by phenylalanine resulted in an enzyme that produced 97% (+)-(1R, 2S)-cis-naphthalene dihydrodiol, in contrast to the wild type, which produced 72% (+)-(1R, 2S)-cis-naphthalene dihydrodiol. This substitution also severely reduced the ability of the enzyme to produce methylcatechols from nitrotoluenes. Instead, the methyl group of each nitrotoluene isomer was preferentially oxidized to form the corresponding nitrobenzyl alcohol. Substitution of a valine at position 258 significantly changed the enantiospecificity of 2NTDO (54% (-)-(1S, 2R)-cis-naphthalene dihydrodiol formed from naphthalene) and the ability of the enzyme to oxidize the aromatic ring of nitrotoluenes. Based on active site modeling using the crystal structure of nitrobenzene 1,2 dioxygenase from Comamonas sp. JS765, Asn258 appears to contribute to substrate specificity through hydrogen bonding to the nitro group of nitrotoluenes.