Glutathione (GSH) is important in antioxidant defense. A major determinant of the rate of GSH synthesis is the activity of the rate-limiting enzyme, γ-glutamylcysteine synthetase (GCS). A heavy (HS) and light subunit (LS) make up GCS; oxidative stress regulates both transcriptionally. Cis-acting elements important for the oxidative stress–induced transcriptional up-regulation of both subunits are antioxidant response element (ARE) and activator protein-1 (AP-1). Nuclear factor-κB (NF-κB) may also regulate the heavy subunit. Chronic ethanol ingestion causes oxidative stress, increases AP-1 expression, and depletes hepatic GSH. Data conflict regarding GSH synthesis and are lacking regarding GCS subunit gene expression. We examined the effect of chronic ethanol ingestion on ARE, AP-1, and NF-κB activity and GCS subunit expression. Male Wistar rats were fed an ethanol and high-fat (28.7% cal) diet intragastrically for 9 weeks. Liver GSH level fell by 40%, although GCS activity doubled. GCS-HS mRNA level doubled, whereas GCS-LS mRNA level remained unchanged. Electrophoretic mobility shift assay (EMSA) showed that binding to ARE, AP-1, and NF-κB probes all increased. In conclusion, chronic ethanol ingestion increased GCS-HS expression and GCS activity by activating cis-acting elements important for transcriptional up-regulation of GCS-HS. GCS-LS mRNA level remained unchanged despite activation of ARE and AP-1, suggesting that negative transcriptional factors may be involved or the mRNA may be unstable. Despite induction in GCS activity, GSH level fell because of alterations in the other factors important in determining the steady-state GSH level.