ABSTRACT.- Nitro-fatty acids (NO2-FAs) are being formed in virgin olive oil (VOO), exhibiting pleiotropic anti-inflammatory responses. We analyzed the fatty acid profile, polyphenols, pigments, and NO2-FA levels in two contrasting olive cultivars, Arbequina and Coratina, under two ripening conditions. During in vitro gastric conditions, the extent of nitro-conjugated linoleic acid and nitro-oleic acid in Coratina VOO was higher at the intermediate stage. In contrast, Arbequina VOO did not present changes on NO2-FA formation. The presence of polyphenols in Arbequina VOO promoted fatty acid nitration, while their absence switched nitration to lipid oxidation processes. In Coratina, the absence of polyphenolic compounds did not affect the NO2-FA levels. This is because Coratina contains greater levels of other bioactive constituents such as pigments that could protect VOO from oxidation. Based on these results, we set the conditions where the composition of VOO promotes the formation of NO2-FA to enhance its health beneficial properties. © 2022 American Chemical Society. All rights reserved.