4-Hydroxy-2-nonenal (HNE) is an aldehyde by-product of lipid peroxidation that is presumed to play a primary role in certain neuropathogenic states (e.g., Alzheimer disease, spinal cord trauma). Although the molecular mechanism of neurotoxicity is unknown, proteomic analyses (e.g., tandem mass spectrometry) have demonstrated that this soft electrophile preferentially forms Michael-type adducts with cysteine sulfhydryl groups. In this study, we characterized HNE synaptosomal toxicity and evaluated the role of putative nucleophilic amino acid targets. Results show that HNE exposure of striatal synaptosomes inhibited (3)H-dopamine membrane transport and vesicular storage. These concentration-dependent effects corresponded to parallel decreases in synaptosomal sulfhydryl content. Calculations of quantum mechanical parameters (softness, electrophilicity) that describe the interactions of an electrophile with its nucleophilic target indicated that the relative softness of HNE was directly related to both the second-order rate constant (k(2)) for sulfhydryl adduct formation and corresponding neurotoxic potency (IC(50)). Computation of additional quantum mechanical parameters that reflect the relative propensity of a nucleophile to interact with a given electrophile (chemical potential, nucleophilicity) indicated that the sulfhydryl thiolate state was the HNE target. In support of this, we showed that the rate of adduct formation was related to pH and that N-acetyl-L-cysteine, but not N-acetyl-L-lysine or beta-alanyl-L-histidine, reduced in vitro HNE neurotoxicity. These data suggest that, like other type 2 alkenes, HNE produces nerve terminal toxicity by forming adducts with sulfhydryl thiolates on proteins involved in neurotransmission.