Following iv administration of 4-hydroxy-2-nonenal (HNE) and [4-3H]HNE to rats, 15 polar urinary metabolites accounting for about 50% of the urinary radioactivity were separated by HPLC. Among them, eight major compounds and tritiated water were quantified. The metabolites were unequivocally characterized using GC/MS and ESI/MS/MS/MS. Most of "HNE polar metabolites" originate from omega-oxidation of 4-hydroxy-2-nonenoic acid (HNA): 9-hydroxy-HNA, its mercapturic acid conjugate, and two diastereoisomers of the corresponding lactone. The oxidation of 9-hydroxy-HNA by alcohol and aldehyde dehydrogenases leads to the excretion of 9-carboxy-HNA and of the corresponding lactone mercapturic acid conjugate. 1, 4-Dihydroxy-2-nonene (DHN) originating from the reduction of HNE by alcohol dehydrogenase was to a lesser extent omega-hydroxylated, leading to 9-hydroxy-DHN which was excreted as a mercapturic acid conjugate (two diastereoisomers).