4-Hydroxy-2-nonenal (HNE) has been recognized as reactive product of lipid peroxidation and has been suggested to play a role in the pathogenesis in several common diseases as well as injuries caused by environmental toxicants. Although formed intracellularly in vivo, for practical reasons this molecule is applied extracellularly in order to analyze its biological effects. The focus of this study was to develop an approach that would enable intracellular HNE production in the absence of the many other products and processes that occur in cells experiencing generalized oxidative stress. To this end, we synthesized 1,1,4-tris(acetyloxy)-2(E)-nonene (HNE[Ac]3), a triester analogue of HNE that is itself unreactive but could be hydrolyzed intracellularly presumably by lipases and/or esterases into the highly reactive HNE. In vitro lipase rapidly converted HNE(Ac)(3) initially to 4-acetyloxy-2-nonenal (HNE[Ac]1) and then to HNE. Neuro 2A cell lysate also caused a rapid hydrolysis of HNE(Ac)3 into HNE(Ac)1 and HNE. Incubation of BSA with HNE(Ac)3 resulted in protein-adduct formation only in the presence of lipase. We demonstrated adduction of HNE to proteins in Neuro 2A cells exposed to HNE(Ac)3 by immunoblotting and immunocytochemistry using antibodies specific for HNE-Michael adducts on proteins. We have previously shown that microtubule organization is very sensitive to HNE. Analysis of Neuro 2A cell microtubules showed that this cytoplasmic organelle is similarly sensitive to HNE and HNE(Ac)3.