Purpose: The products of lipid peroxidation have been implicated in human diseases and aging. This prompted us to investigate the response to conventional (CONV) versus FLASH irradiation of oxylipins, a family of bioactive lipid metabolites derived from omega-3 or omega-6 polyunsaturated fatty acids through oxygen-dependent non-enzymatic as well as dioxygenase-mediated free radical reactions. Methods and Materials: Ultrahigh performance liquid chromatography coupled to tandem mass spectrometry was used to quantify the expression of 37 oxylipins derived from eicosatetraenoic, eicosapentaenoic and docosahexaenoic acid in mouse lung and in normal or cancer cells exposed to either radiation modality under precise monitoring of the temperature and oxygenation. Among the 37 isomers assayed, 14-16 were present in high enough amount to enable quantitative analysis. The endpoints were the expression of oxylipins as a function of the dose of radiation, normoxia versus hypoxia, temperature and post irradiation time. Results: In normal, normoxic cells at 37 degrees C radiation elicited destruction and neosynthesis of oxylipins acting antagonistically on a background subject to rapid remodeling by oxygenases. Neosynthesis was observed in the CONV mode only, in such way that the level of oxylipins at 5 minutes after FLASH irradiation was 20-50% lower than in non-irradiated and CONV-irradiated cells. Hypoxia mitigated the differential CONV versus FLASH response in some oxylipins. These patterns were not reproduced in tumor cells. Depression of specific fi c oxylipins following FLASH irradiation was observed in mouse lung at 5 min following irradiation, with near complete recovery in 24 hours and further remodeling at one week and two months post irradiation. Conclusions: Down-regulation of oxylipins was a hallmark of FLASH irradiation specific fi c of normal cells. Temperature effects suggest that this process occurs via diffusion-controlled, bimolecular recombination of a primary radical species upstream from peroxyl radical formation and evoke a major role of the membrane composition and fl uidity in response to the FLASH modality. (c) 2024 Elsevier Inc. All rights reserved.