Technical Abstract: Alzheimer’s disease elevates lipid peroxidation in the brain and data indicate that lipid-aldehydes are pathological effectors of lipid peroxidation. The disposition of 4-substituted nonenals derived from arachidonate (20:4, n-6) and linoleate (18:2, n-6) oxidation is modulated by their protein adduction targets, metabolism, and the nature of the 4-substitutent. Trans-4-oxo-2-nonenal (4-ONE) has a higher chemical reactivity in some systems than the more commonly known trans-4-hydroxy-2-nonenal (HNE). In the present work, we performed a structure-function analysis of 4-hydroxy/oxo-alkenal upon mitochondrial endpoints. We tested the hypotheses that 4-ONE, owing to a highly reactive nature, is more toxic than HNE and that HNE toxicity is enantioselective. We chose to study freshly isolated brain mitochondria because of the role of mitochondrial dysfunction in neurodegenerative disorders. While there was little effect related HNE chirality, our data indicate that in the mitochondrial environment, the order of toxic potency under most conditions was 4-ONE> HNE. 4-ONE uncoupled mitochondrial respiration at a concentration of 5 µM and inhibited ALDH2 activity with an IC50 of approximately 0.5 µM. The efficacy of altering mitochondrial endpoints were ALDH2 inhibition > respiration = mitochondrial swelling = ALDH5A inhibition > GSH depletion. Thiol-based alkenal scavengers, but not amine-based scavengers, were effective in blocking the effects of 4-ONE upon respiration. Quantum mechanical calculations provided insights into the basis for the elevated reactivity of 4-ONE > HNE. Our data demonstrate that 4-ONE is a potent effector of lipid peroxidation in the mitochondrial environment.