Trans-4-hydroxy-2-nonenal (HNE) is a product of lipid peroxidation with many cellular effects. HNE possesses a stereogenic center at the C4 carbon that influences the metabolism and alkylation targets of HNE. We tested the hypothesis that rat brain mitochondria metabolize HNE in an enantioselective manner after exposure to racemic HNE. The study of HNE chirality, however, is hindered by the lack of facile methods to chromatographically resolve (R)-HNE and (S)-HNE. We used a chiral hydrazine, (S)-carbidopa, as a derivatization reagent to form diastereomers with (R)-HNE and (S)-HNE that were separated by reverse-phase HPLC. After exposure to racemic HNE, rat brain mitochondria metabolized HNE enantioselectively with a higher rate of (R)-HNE metabolism. By using the purified enantiomers of HNE, we found that this enantioselective metabolism of HNE was the result of higher rates of enzymatic oxidation of (R)-HNE by aldehyde dehydrogenases compared to (S)-HNE. Conjugation of HNE to glutathione was a minor metabolic pathway and was not enantioselective. These studies demonstrate that the chirality of HNE affects its mitochondrial metabolism and potentially other processes in the central nervous system.