High expression of the aflatoxin B1 (AFB1)-8,9-epoxide-conjugating glutathione S-transferase A3 (mGSTA3) subunit in mouse liver confers intrinsic resistance to AFB1 hepatocarcinogenesis. It is not known how the gene encoding this protein is regulated. The murine mGSTA3 gene has been identified using bioinformatics. It localizes to mouse chromosome 1 (A3-4), spans approximately 24.6 kilobases (kb) of DNA, and comprises seven exons. High levels of mGSTA3 mRNA are present in organs associated with detoxification. Expression of mGSTA3 in Hepa1c1c7 mouse hepatoma cells was found to be inducible by sulforaphane, an organic isothiocyanate that can transcriptionally activate genes through the antioxidant response element (ARE). Sulforaphane also induced transcription of a luciferase reporter containing a 1.5 kb fragment of the mGSTA3 5'-upstream region. A putative ARE, with sequence 5'-TGACATTGC-3', was identified within this fragment, approximately 150 base pairs upstream of exon 1. Mutation of this sequence abrogated both basal and sulforaphane-inducible reporter activity. Overexpression of the basic-region leucine zipper Nrf2 transcription factor augmented activity of the mGSTA3-luciferase reporter through this ARE. Electrophoretic mobility shift assays demonstrated that Nrf2 binds the mGSTA3 ARE. Measurement of mGSTA3 mRNA levels in tissues isolated from both wild-type and nrf2-null mice revealed that loss of the Nrf2 transcription factor is associated with a reduction in basal expression of mGSTA3. Collectively, these data demonstrate a role for Nrf2 and the ARE in regulating transcription of mGSTA3.