The structural modifications of the unsaturated fatty acid components of triglycerides in extra virgin olive oil (EVOO) following exposure to nitrite ions in acidic media were determined by two-dimensional (2D) NMR spectroscopy, aided by (15)N labeling and GC analysis, allowing investigation of the matrix without fractionation steps. In the presence of excess nitrite ions in a 1% sulfuric acid/oil biphasic system, extensive double bond isomerization of the oleic/linoleic acid components of triglycerides was observed associated with nitration/oxidation processes. Structurally modified species were identified as E/Z-nitroalkene, 1,2-nitrohydroxy, and 3-nitro-1-alkene(1,5-diene) derivatives based on (1)H, (13)C, and (15)N 2D NMR analysis in comparison with model compounds. Minor constituents of EVOO, including phenolic compounds and tocopherols, were also substantially modified by nitrite-derived nitrating species, even under milder reaction conditions relevant to those occurring in the gastric compartments. Novel nitrated derivatives of tyrosol, hydroxytyrosol, and oleuropein (6-8) were identified by LC/MS analysis of the polar fraction of EVOO and by comparison with synthetic samples. Overall, these results provide the first systematic description at the chemical level of the consequences of exposing EVOO to nitrite ions at acidic pH and offer an improved basis for further investigations in the field of toxic nitrosation/nitration reactions and dietary antinitrosating agents.