Three 2,4,6-trinitrotoluene (TNT) nitroreductases from Klebsiella sp. C1 have different reduction capabilities that can degrade TNT by simultaneous utilization of two initial reduction pathways. Of these, nitroreductase II was purified to homogeneity by sequential chromatographies. Nitroreductase II is an oxygeninsensitive enzyme and reduces both TNT and nitroblue tetrazolium. The N-terminal amino acid sequence of the enzyme did not show any sequence similarity with those of other nitroreductases reported. However, it transformed TNT by the reduction of nitro groups like nitroreduclase I. It had a higher substrate affinity and specific activity for TNT reduction than other nitroreductases, and it showed a higher oxidation rate of NADPH with the ortho-substituted isomers of TNT metabolites (2-hydroxylaminodinitrotoluene and 2-aminodinitrotoluene) than with para-substituted compounds (4-hydroxylaminodinitrotoluene and 4-aminodinitrotoluene).