Denitration of 2,4,6-trinitrotoluene (TNT) was evaluated in oxygen-depleted enrichment cultures. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum and contained TNT as sole nitrogen source. Incubations were carried out in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite, and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. In addition to reduced derivatives of TNT, several unidentified metabolites were detected. Concomitant to a decrease of TNT concentration, a release of nitrite was observed. The concentration of nitrite peaked and then it slowly decreased. In the absence of TNT, the drop in the concentration of nitrite in oxygen-depleted cultures was lower when ferrihydrite was provided, suggesting that ferrihydrite inhibited the utilization of nitrite by P. aeruginosa ESA-5.