Many microbes reduce the nitro substituents of 2,4,6-trinitrotoluene (TNT), producing aminodinitrotoluenes (ADNTs). These compounds are recalcitrant to further breakdown and are acutely toxic. In a search for organisms capable of metabolizing ADNTs, a bacterial strain was isolated for the ability to use 2-aminobenzoate (anthranilate) as sole C-source. This isolate, Pseudomonas aeruginosa MA01, metabolized TNT by first reducing one nitro group to form either 2-amino-4,6-dinitrotoluene (2ADNT) or 4-amino-2,6-dinitrotoluene (4ADNT). However, strain MA01 was distinct from other TNT-reducing organisms in that it transformed these compounds into highly polar metabolites through an O2-dependent process. Strain MA01 was able to cometabolize TNT, 2ADNT, and 4ADNT in the presence of a variety of carbon and energy sources. During aerobic cometabolism with succinate, 45% of uniformly ring-labeled [14C]TNT was transformed to highly polar compounds. Aerobic cometabolism of purified [14C]2ADNT and [14C]4ADNT with succinate as C-source produced similar amounts of these polar metabolites. During O2-limited cometabolism with succinate as C-source and nitrate as electron acceptor, less than 8% of the [14C]TNT was transformed to polar metabolites. Purified 2,6-diamino-4-nitrotoluene was not metabolized, and while 2,4-diamino-6-nitrotoluene was acetylated, the product (N-acetyl-2,4-diamino-6-nitrotoluene) was not further metabolized. Therefore, strain MA01 metabolized TNT by oxidation of the ADNTs and not by reduction the remaining nitro groups on the ADNTs.