We have investigated the ability of apoE (apolipoprotein E) to participate in the biogenesis of HDL (high-density lipoprotein) particles in vivo using adenovirus-mediated gene transfer in apoA-I^−/− (apolipoprotein A-I) or ABCA1^−/− (ATP-binding cassette A1) mice. Infection of apoA-I^−/− mice with 2×10⁹ pfu (plaque-forming units) of an apoE4-expressing adenovirus increased both HDL and the triacylglycerol-rich VLDL (very-low-density lipoprotein)/IDL (intermediate-density lipoprotein)/LDL (low-density lipoprotein) fraction and generated discoidal HDL particles. ABCA1^−/− mice treated similarly failed to form HDL particles, suggesting that ABCA1 is essential for the generation of apoE-containing HDL. Combined infection of apoA-I^−/− mice with a mixture of adenoviruses expressing both apoE4 (2×10⁹ pfu) and human LCAT (lecithin:cholesterol acyltransferase) (5×10⁸ pfu) cleared the triacylglycerol-rich lipoproteins, increased HDL and converted the discoidal HDL into spherical HDL. Similarly, co-infection of apoE^−/− mice with apoE4 and human LCAT corrected the hypercholesterolaemia and generated spherical particles, suggesting that LCAT is essential for the maturation of apoE-containing HDL. Overall, the findings indicate that apoE has a dual functionality. In addition to its documented functions in the clearance of triacylglycerol-rich lipoproteins, it participates in the biogenesis of HDL-sized apoE-containing particles. HDL particles generated by this pathway may account at least for some of the atheroprotective functions of apoE.