This study was undertaken to identify the alpha-helical domains of human apoE that mediate cellular cholesterol efflux and HDL assembly via ATP-binding cassette transporter A1 (ABCA1). The C-terminal (CT) domain (residues 222-299) of apoE was found to stimulate ABCA1-dependent cholesterol efflux in a manner similar to that of intact apoE2, -E3, and -E4 in studies using J774 macrophages and HeLa cells. The N-terminal (NT) four-helix bundle domain (residues 1-191) was a relatively poor mediator of cholesterol efflux. On a per molecule basis, the CT domain stimulated cholesterol efflux with the same efficiency (Km approximately 0.2 microM) as intact apoA-I and apoE. Gel filtration chromatography of conditioned medium from ABCA1-expressing J774 cells revealed that, like the intact apoE isoforms, the CT domain promoted the assembly of HDL particles with diameters of 8 and 13 nm. Removal of the CT domain abolished the formation of HDL-sized particles, and only larger particles eluting in the void volume were formed. Studies with CT truncation mutants of apoE3 and peptides indicated that hydrophobic helical segments governed the efficiency of cellular cholesterol efflux and that conjoined class A and G amphipathic alpha-helices were required for optimal efflux activity. Collectively, the data suggest that the CT lipid-binding domain of apoE encompassing amino acids 222-299 is necessary and sufficient for mediating ABCA1 lipid efflux and HDL particle assembly.