Transcriptional activation of the mouse glutathione S-transferase Ya gene by chemoprotective molecules is mediated through the interaction of trans-acting factors with an antioxidant responsive element (ARE) in the promoter region of this gene. In a step toward identifying those factors which bind productively to the GST Ya ARE, all of the discernible, specific ARE-binding proteins (ARE-BP) in nuclear extracts from HepG2 cells were systematically characterized. By gel-mobility-shift analysis, seven specific ARE-BPs, termed ARE-BP-1 through 7 in order of increasing mobility, were observed that did not vary in concentration or migration between induced and uninduced cell extracts. The molecular weights of the individual ARE-BP subunits were determined by a two-dimensional electrophoresis protocol. Ferguson gel analysis of native protein size indicated that several of the ARE-BP-DNA complexes are composed of multiple protein subunits. Wild-type AREs and GST Ya ARE fragments and mutant sequences were evaluated for their ability to mediate induction in a reporter gene system in HepG2 cells. This same panel of sites was tested in an in vitro binding assay for the ability to compete for the ARE-BPs. A binding profile for each ARE-BP was compiled. Correlation between the ARE-BP binding profiles and induction results indicated that: (i) the ARE-BP-1 and ARE-BP-2 complexes formed only with AREs that supported induction, and (ii) the ARE-BP-4 complex formed with all inducible AREs, but it also bound to ARE mutants that failed to support induction. Based on the studies, an early composite regulatory element model for ARE-mediated expression is presented. ARE-BP-1 is proposed to be the mediator of the ARE's unique induction response to chemoprotective agents.