α,β-Unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE) and other electrophilic lipid peroxidation (LPO) products may contribute to the pathogenesis of cancer, cardiovascular diseases, and other age-related diseases by cytotoxic, genotoxic, and proinflammatory mechanisms. The notion that vitamin C (ascorbic acid) acts as a biological antioxidant has been challenged recently by an in vitro study showing that ascorbic acid promotes, rather than inhibits, the formation of genotoxic LPO products from the lipid hydroperoxide, hydroperoxy octadecadienoic acid [Lee, S. H., Oe, T. & Blair, I. A. (2001) Science 292, 2083–2086]. Here, we demonstrate that ascorbic acid acts as a nucleophile and forms Michael-type conjugates with electrophilic LPO products. Several ascorbyl-LPO product conjugates, resulting from the interaction of ascorbic acid with hydroperoxy octadecadienoic acid in vitro, were identified by tandem MS, including ascorbyl conjugates of HNE, 4-oxo-2-nonenal, and presumably, 12-oxo-9-hydroxy-10-dodecenoic acid. The same ascorbyl-LPO product conjugates were detected in human plasma. The concentration of the ascorbyl-HNE conjugate in plasma from 11 healthy subjects was found to be 1.30 ± 0.74 μM (mean ± SD). Our data identify ascorbylation (vitamin C conjugation) as a previously unrecognized, biologically relevant pathway for the elimination of electrophilic LPO products, and have implications for the prevention and treatment of chronic inflammatory diseases, as well as the development of novel biomarkers of oxidative stress