Enterobacter cloacae strain 96-3 nitroreductase (NR) is a homodimeric flavoenzyme that catalyzes the pyridine nucleotide-dependent four-electron reduction of a variety of nitroaromatic compounds, including the explosives TNT (2,4,6-trinitrotoluene), RDX (1,3,5-trinitro-1,3,5-triazine), tetryl (2,4,6-trinitrophenyl-N-methylnitramine), and pentryl (2,4,6-trinitrophenyl-N-nitroaminoethylnitrate). The enzyme was initially characterized by Bryant et al. from a strain of Enterobacter that had been isolated from a weapons dump in La Jolla, CA. The enzyme displays a catalytic efficiency for nitroreduction at least 10-fold higher than that of several highly homologous bacterial nitroreductases and has long been thought to have evolved to be a more efficient nitroreductase due to the high nitroaromatic compound concentrations in its environment. We report the cloning and biochemical characterization of a nitroreductase gene from a clinical isolate of Enterobacter cloacae, a strain that presumably had not encountered high concentrations of nitroaromatics. The new enzyme, which we term retro-nitroreductase, had an amino acid sequence 96.7% identical to NR, and most differences are relatively conservative. The catalytic efficiency of the new enzyme is twofold less than that of NR for the oxidation of NADH and is not significantly different from the value observed for NR for the reduction of dinitrobenzyl alcohol. We conclude that NR has not significantly evolved to be a more efficient nitroreductase as a result of its environment, and the relatively high catalytic activity of the enzyme is a general property of Enterobacter cloacae nitroreductases.