Enhanced oxidative stress is implicated in the development of atherosclerosis in humans and animal models. F2-isoprostanes are formed in vivo via free radical peroxidation of arachidonic acid, and their quantification has allowed assessment of oxidative stress in vivo. F2-isoprostanes associate with lipids, although their distribution in human plasma lipoproteins is unknown. Our aim was to determine the distribution and levels of F2-isoprostanes in lipoproteins isolated from human plasma by ultracentrifugation and fast protein liquid chromatography (FPLC). F2-isoprostanes were significantly higher in HDL compared with LDL or VLDL after isolation by ultracentrifugation or FPLC. Furthermore, HDL3 particles contained elevated levels of F2-isoprostanes compared with HDL2. Platelet activating factor acetylhydrolase (PAF-AH), which hydrolyses esterified F2-isoprostanes from phospholipids, was predominantly associated with LDL. Reduced F2-isoprostanes in LDL may be related to higher PAF-AH activity in LDL. Paraoxonase 1 (PON-1) activity was associated with HDL2 and may be a contributing factor to the lower F2-isoprostanes in HDL2 compared with HDL3. Further studies are required to establish the implications of these findings on HDL function.