Analysis of the reaction between 2'-deoxyadenosine and 4-oxo-2-nonenal by liquid chromatography/mass spectrometry revealed the presence of three major products (adducts A1, A1, and B). Adducts A1 and A1 were isomeric; they interconverted at room temperature, and they each readily dehydrated to form adduct B. The mass spectral characteristics of adduct B obtained by collision-induced dissociation coupled with multiple tandem mass spectrometry were consistent with those expected for a substituted etheno adduct. The structure of adduct B was shown by NMR spectroscopy to be consistent with the substituted etheno-2'-deoxyadenosine adduct 1''-[3-(2'-deoxy-β-D- erythropentafuranosyl)-3H-imidazo[2,1-i]purin-7-yl]heptane-2''-one. Unequivocal proof of structure came from the reaction of adducts A1 and A2 (precursors of adduct B) with sodium borohydride. Adducts A2 and A2 each formed the same reduction product, which contained eight additional hydrogen atoms. The mass spectral characteristics of this reduction product established that the exocyclic amino group (N6) of 2'-deoxyadenosine was attached to C-1 of the 4-oxo-2-nonenal. The reaction of 4-oxo-2-nonenal with calf thymus DNA was also shown to result in the formation of substituted etheno adducts A1 and A1 and substituted etheno adduct B. Adduct B was formed in amounts almost 2 orders of magnitude greater than those of adducts A1 and A1. This was in keeping with the observed stability of the adducts. The study presented here has provided additional evidence which shows that 4- oxo-2-nonenal reacts efficiently with DNA to form substituted etheno adducts.