Acrolein, 3-oxopropyl glutathione (oxoPrGSH), andS-3-hydroxypropylN-acetylcysteine (hydroxyPrMCA) are confirmed metabolic products of cyclophosphamide. Other potential metabolites include the mercapturic acidS-3-oxopropylN-acetylcysteine (oxo- PrMCA), its sulfoxide, and their corresponding diacid forms. The reactivity of acrolein would appear to preclude its movement from the main site of formation (liver) to the sites of toxicity (lung and bladder). However, the rerelease of acrolein from various thiol conjugates via a β-elimination reaction is possible. It is also possible that the parent conjugate is directly toxic. The current study examined the toxicity of various acrolein–thiol conjugates and related analogs to human lung adenoma A549 cells. The expected enhancement of acrolein, oxoPrMCA, and oxoPrMCAS-oxide toxicity (assessed as cell proliferation by the alamarBlue assay) following a 2-hr exposure of cells treated with diethyl maleate (DEM) to deplete GSH was observed. OxoPrGSH was toxic when present for 24 hr, and this toxicity was also enhanced by pretreatment with DEM. When treated with these conjugates alone, the depletion of intracellular GSH only occurred at doses above those needed to significantly inhibit cell proliferation. There were no changes in protein thiols as determined using the membrane impermeant fluorescent thiol probepara-sulfobenzoyloxybromobimane. The diacid conjugate was not toxic to A549 cells indicating further oxidation products of the mercapturic acids are not a factor in toxicity. ButanoneMCA, an analog of oxoPrMCA that cannot exist in the geminal diol form, inhibited A549 cell growth only slightly less effectively than oxoPrMCA, suggesting the geminal diol is not toxicologically significant. OxoBuMCA, which cannot undergo β-elimination of acrolein, showed no toxicity to these cells, suggesting that the release of acrolein could be required. Exogenous GSH provided protection from all toxic compounds suggesting that the toxic species is electrophilic. Overall, the data suggest that toxicity, in terms of an inhibition of cell proliferation, is not due to the parent molecule but rather is the result of rereleased acrolein that then affects factors necessary for cell proliferation.