Previous studies from this laboratory demonstrated that 4-hydroxy-2-nonenal (4HNE), a lipid peroxidation product, induces expression of γ-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo glutathione (GSH) synthesis, in rat alveolar epithelial L2 cells. The present study demonstrates that 4HNE also induces GCS in primary cultured alveolar epithelial type II (AT2) cells. Enzyme activity, protein content, and messenger RNA levels of both the catalytic (GCS-HS) and regulatory (GCS-LS) subunits were significantly increased in AT2 cells treated with 5 or 10 μM 4HNE, the same concentrations that induced GCS expression in L2 cells. As in L2 cells, 4HNE induced a greater AT2-cell increase in GCS-LS than in GCS-HS, suggesting that modulation of GCS-LS may play a dominant role in regulating GSH concentration in response to oxidative stress. Additional studies using mitogen-activated protein kinase pathway inhibitors showed that induction by 4HNE of GCS-HS, but not GCS-LS, was mediated through activation of the extracellular regulated kinase pathway in L2 cells. The results demonstrate that L2 cells maintain the same responsiveness to oxidant challenge as do primary cultured AT2 cells in terms of increasing GSH synthetic capacity, and that different pathways are involved in the induction of two GCS subunits by 4HNE.