We investigated the formation of F2-isoprostanes produced by non-enzymatic peroxidation of arachidonic acid during rabbit aortic endothelial cell-mediated oxidation of low density lipoprotein (LDL). Free and total (sum of free and esterified) levels of F2-isoprostanes were measured using a solid-phase extraction procedure and gas chromatography-mass spectrometry. Free levels of F2-isoprostanes in native LDL were 0.06 +/- 0.03 ng/mg protein (n = 4), whereas total levels were 0.28 +/- 0.09 ng/mg protein (n = 4). Both free and total levels of the isoprostanes were found to increase during the oxidation. 8-epi-PGF2 alpha was the major isoprostane formed (free and total concentrations after 24 h, 2.50 +/- 0.24 and 6.42 +/- 1.36 ng/mg protein (n = 4), respectively). The release of F2-isoprostanes during aortic endothelial cell-induced oxidation of LDL could be a contributory factor in the development of atherosclerosis.