The exact role of azodye reductase, a liver microsmal enzyme, and its influence on the hepatocarcinogenicity of 4-dimethylaminoazobenzene (DAB) are uncertain. The effect of DAB is depressed by many nutritional factors, including trytophan. Therefore, the effects of o-aminophenol and of L-tryptophan and its metabolites L-kynurenine, anthranilic acid, kynurenic acid, quinaldic acid, 3-hydroxy-DL-kynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, N-methylnicotinamide, and N′-methylnicotin-amide on rat liver azoreductase activity were determined, using DAB as substrate. Only 3-hydroxyanthran- ilic acid, 3-hydroxykynurenine and o-aminophenol decreased enzyme activity. The inhibition was greater if the buffered solutions (pH 7.4) of these three compounds were kept overnight before use, but the effect was prevented if these compounds were prepared in solutions of L-ascorbic acid and/or L-cysteine HCl. This observation indicates that the autoxidation products were probably responsible for inhibition of the enzyme. Further study of the oxidation products including the phenylquinoneimine formed from the oxidation of 3-hydroxyanthranilic acid in air, cinnabarinic acid, xanthommatin, 2-amino-3H-isophenoxazin-3-one, 1,9-dimethyl-2-amino-3H-phenoxazin-3-one and actinomycin D showed that all these compounds inhibited enzyme activity. A non-competitive type of inhibition was observed in the presence of cinnabarinic acid and xanthommatin. Cinnabarinic acid, xanthommatin, 2-amino-3H-isophenoxazin-3-one, 1,9-dimethyl-2-amino-3H-phenoxazin-3-one, a nd actinomycin D all have the same phenoxazinone ring system, suggesting that the driving factor in the inhibition of the azoreductase is the presence of the phenoxazinone chromophore. The chemical resemblance of these phenoxazinones to the coenzyme riboflavine further supports this supposition.