Exposure of HepG2 cells to beta-naphthoflavone (beta-NF) results in time- and dose-dependent increase in the steady-state mRNA levels for both the catalytic (GCSh) and regulatory (GCS1) subunits of gamma-glutamylcysteine synthetase (GCS) which catalyzes the rate-limiting step in the de novo synthesis of the cellular antioxidant glutathione (GSH) (Mulcahy, R. T., Wartman, M. A., Bailey, H. B., and Gipp, J. J. (1997) J. Biol. Chem. 272, 7445-7454). Cloning and sequencing of the GCS1 promoter region is reported. Regulatory sequences mediating basal and beta-NF induced expression of the GCSl gene were identified using a series of promoter/reporter fusion genes transfected into HepG2 cells. Sequences directing basal and beta-NF induced expression were localized between nucleotides -344 and -242 (numbered relative to the translation start site). Mutational analyses indicate that basal expression of the GCSl gene is directed by a consensus AP-1-binding site located 33 base pairs upstream of a consensus electrophile responsive element (EpRE) sequence; both cis-elements are capable of supporting beta-NF inducibility. Elimination of the inducible response requires simultaneous mutation of both sequences, however, in the presence of an intact EpRE the upstream AP-1 site is irrelevant to induction. Regulation of expression of both human GCS subunit genes in response to beta-NF is therefore mediated by cis-elements satisfying the consensus core EpRE motif.