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The plasma HDLs represent a major class of cholesterol-transporting lipoprotein that can be divided into two distinct subfractions, HDL2 and HDL3, by ultracentrifugation. Existing methods for the subfractionation of HDL requires lengthy ultracentrifugations, making them unappealing for large-scale studies. We describe a method that subfractionates HDL from plasma in only 6 h, representing a substantial decrease in total isolation time. The subfractions so isolated were assessed for a variety of lipid and protein components, in addition to their susceptibility to oxidation, both alone and in combination with VLDL and LDL. We report for the first time a prooxidant role for HDL during VLDL oxidation, in which HDL donates preformed hydroperoxides to VLDL in a cholesteryl ester transfer protein (CETP)-dependent process. Examination of the participation of HDL in LDL oxidation has reinforced its classic role as a potent antioxidant. Furthermore, we have also implicated the second major HDL-associated enzyme, LCAT, in these processes, whereby it acts as a potent prooxidant during VLDL oxidation but as an antioxidant during LDL oxidation. Thus, we have identified a potentially duplicitous role for HDL in the pathogenesis of atherosclerosis, attributable to both CETP and LCAT.