A nuclear protein from HepG2 cells (YABP) that binds the antioxidant response element (ARE), which is required for activation of the rat glutathione S-transferase (GST) Ya subunit gene by planar aromatic compounds and phenolic antioxidants, was further characterized by quantitative competition binding experiments and DNA mutational analysis. The apparent dissociation constant of the YABP-ARE complex was estimated as < or = 0.77 nM, suggesting that the YABP has very high affinity for the ARE. There is no difference in the affinity of the YABP for the ARE when HepG2 cells are treated with inducers that transcriptionally activate the GST Ya subunit gene. Quantitative competition binding analyses in conjunction with mutagenesis of the ARE revealed that an 11-nucleotide region in the 41-nucleotide ARE, 5'-GGTGACAAAGC-3', is responsible for binding to the YABP. Eight nucleotides of this core sequence are in close proximity to the YABP, indicating that there is a broader spectrum of protein contact points than those required for the transcriptional activation. van't Hoff analysis of effects of temperature on binding has revealed that the binding reaction is governed mainly by entropy changes, which could result from conformational changes in the YABP and/or the ARE upon the formation of the complex. In addition, the native molecular weight of the YABP was determined to be 74 300 using gel filtration chromatography. These data together with previous UV cross-linking data suggest that the YABP exists as a heterodimer.