The mechanism of acrolein-induced lipid peroxidation is unknown. This study found that acrolein and its glutathione adduct, glutathionylpropionaldehyde, induce oxygen radical formation. These oxygen radicals may be responsible for the induction of lipid peroxidation by acrolein. The enzymes xanthine oxidase and aldehyde dehydrogenase were found to interact with glutathionylpropionaldehyde to produce O₂^.− and HO^.. Acrolein was oxidized by xanthine oxidase to produce acroleinyl radical and O₂^.−. Aldehyde dehydrogenase metabolized acrolein to form O₂^.− but not acroleinyl radical. The fact that glutathionylpropionaldehyde is a more potent stimulator of oxygen radical formation than acrolein indicates that glutathionylpropionaldehyde is a toxic metabolite of acrolein and may be responsible for some of the in vivo toxicity of acrolein.