In patients with COPD an imbalance between oxidants and anti-oxidants is acknowledged to result in disease development and progression. Cigarette smoke (CS) is known to deplete total glutathione (GSH+GSSG) in the airways. We hypothesized that components in the gaseous-phase of CS may irreversibly react with GSH to GSX, thereby causing this depletion. To understand this phenomenon, we investigated the GSH metabolism in response to CS, and identified the actual GSX compounds. Both CS and H₂O₂-control depletes reduced GSH in solution ( -54.1±1.7 µM, P<0.01 and -39.8±0.9 µM; p<0.01). However, a significant decrease of total glutathione was observed after CS exposure ( -75.1±7.6 µM, P<0.01) but not after H₂O₂ exposure. Exposure of A549 cells and primary bronchial epithelial cells to CS decreased free sulfhydryl (-SH) groups ( -64.2±14.6 µM/mg protein; p<0.05) and irreversibly modified total glutathione ( -17.7±1.9 µM; p<0.01) compared to non-exposed cells or H₂O₂ control. Mass spectrometry (MS) showed that GSH was modified into glutathion-aldehyde derivatives. Further MS identification showed that GSH was bound to acrolein and crotonaldehyde, and another, yet unidentified structure. Our data shows that CS did not oxidize GSH to GSSG, but reacts to non-reducible glutathione-aldehyde derivatives, thereby depleting the total available GSH-pool.