Reductive metabolism of aromatic nitro compounds was examined with rabbit liver preparations. Under anaerobic conditions, carcinogenic 2-nitrofluorene, 4-nitrobiphenyl, and 1-nitro-naphthalene were reduced to the corresponding hydroxylamines and amines, whereas the carcinogenic 1-nitropyrene was reduced only to the corresponding amine by liver cytosol in the presence of 2-hydroxypyrimidine, an electron donor of aldehyde oxidase. These metabolites were identified unequivocally by comparing their mass spectra and thin-layer chromatographic behaviors with those of the authentic samples. Both liver microsomes and cytosol catalyzed the reduction of these aromatic nitro compounds in varying degrees. The microsomes required reduced pyridine nucleotides for occurrence of the nitroreductase activities. In this case, reduced nicotinamide adenine dinucleotide phosphate was more effective than reduced nicotinamide adenine dinucleotide as an electron donor. The cytosol by itself exhibited some nitroreductase activities, which were markedly enhanced by addition of an electron donor of aldehyde oxidase, i.e., N1-methylnicotinamide or 2-hydroxypyrimidine. The full activities of the cytosol with the electron donor were much higher than those of the microsomes with the reduced pyridine nucleotide. Purified liver aldehyde oxidase, like the cytosol, exhibited significant nitroreductase activities in the presence of its electron donor. These results indicated that cytosolic aldehyde oxidase functions as a major enzyme responsible for the reduction of aromatic nitro compounds including carcinogens in rabbit liver.