Antioxidant response elements (AREs) containing 12-O-tetradecanoylphorbol-13-acetate response element (TRE) (perfect AP1) and TRE-like (imperfect AP1) elements mediate high basal transcription of the NAD(P)H:quinone oxidoreductase1 (NQO1) and glutathione S-transferase Ya genes in tumor cells and its induction in response to xenobiotics and antioxidants. Mutations in the human NQO1 gene ARE (hARE) revealed the requirement for two TRE or TRE-like elements arranged in inverse orientation at the interval of three base pairs and a GC box for optimal expression and beta-naphthoflavone induction of the NQO1 gene. A single TRE element from the human collagenase gene failed to respond to beta-naphthoflavone. These results demonstrate that ARE (2 x TRE or TRE-like elements)-containing detoxifying enzyme genes and not genes that contain 1 x TRE are responsive to xenobiotics and antioxidants. Bandshift assays showed shifting of a complex of more or less similar mobility with hARE and TRE that could be competed by each other. Mutations in the 3'-TRE of the NQO1 gene hARE eliminated binding of nuclear proteins to the hARE and resulted in the loss of basal and induced expression, indicating that 3'-TRE is the most important element within the hARE. 5'-TRE-like element within the NQO1 gene hARE is required for xenobiotic response but may not bind to the nuclear proteins by itself. The GC box located immediately following the 3'-TRE is required for optimal expression and induction of the NQO1 gene. The comparison of AREs from several different genes indicated the requirement for specific arrangement and spacing of two TRE and TRE-like elements within the AREs.