4-Hydroxy-2-nonenal (HNE) is an endogenous product of lipid peroxidation, which is believed to play a biological role in the pathogenesis of various diseases. HNE is formed as a racemic mixture of (R)- and (S)- enantiomers. These enantiomers differ in their biological properties. The aim of this study was to investigate separately the in vivo metabolism of the two HNE enantiomers in male rats after intravenous administration of the corresponding radiolabeled compounds and to compare the results with those obtained with the racemic mixture. Although the difference in the excretion rates was not statistically significant, the HPLC profiles of urinary metabolites showed qualitative and quantitative differences between the two enantiomers. The level of 3-mercapturic acid-1,4-dihydroxynonane, which is considered as the major urinary metabolite of HNE, was significantly lower in the case of (S)-HNE injected rats. In vitro studies using rat liver cytosolic incubations and HNE-glutathione conjugate as substrate were performed to clarify the intermediate pathways involved in their metabolism. Large differences were obtained in the reduction and retro-Michael conversion steps of the metabolism between the conjugates originating from the two enantiomers.