Glutamate-cysteine ligase is a rate-limiting enzyme in the de novo synthesis of glutathione, a known scavenger of electrophiles and reactive oxygen species. Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by nuclear factor erythroid 2-related factor 2 (Nrf2). It has been reported that ethanol induces human GCLC production via Nrf2-mediated transactivation of the antioxidant-responsive element (ARE). Here, the luciferase reporter assay revealed the presence of an ethanol-responsive element in the human GCLC promoter; it spanned bases -1432 to -832 in hepatocytes and HepG2 cells transfected with cytochrome P450 2E1 (CYP2E1). The region lacked an ARE but had a putative nuclear factor-kappaB (NF-kappaB) element. NF-kappaB DNA-binding activity was activated in response to ethanol treatment. CYP2E1 expression was required for GCLC promoter-driven gene expression and the activation of NF-kappaB. Thus ethanol-induced GCLC transcription is mediated by not only Nrf2 but also NF-kappaB.