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Apolipoprotein E (apoE)/ABCA1 interactions were investigated in human intact fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Here, we show that purified human plasma apoE3 forms a complex with ABCA1 in normal fibroblasts. Lipid-free apoE3 inhibited the binding of 125I-apoA-I to ABCA1 more efficiently than reconstituted HDL particles (IC50 = 2.5 ± 0.4 μg/ml vs. 12.3 ± 1.3 μg/ml). ApoE isoforms showed similar binding for ABCA1 and exhibited identical kinetics in their abilities to induce ABCA1-dependent cholesterol efflux. Mutation of ABCA1 associated with Tangier disease (C1477R) abolished both apoE3 binding and apoE3-mediated cholesterol efflux. Analysis of apoE3-containing particles generated during the incubation of lipid-free apoE3 with stimulated normal cells showed nascent apoE3/cholesterol/phospholipid complexes that exhibited preβ-electrophoretic mobility with a particle size ranging from 9 to 15 nm, whereas lipid-free apoE3 incubated with ABCA1 mutant (C1477R) cells was unable to form such particles.These results demonstrate that 1) apoE association with lipids reduced its ability to interact with ABCA1; 2) apoE isoforms did not affect apoE binding to ABCA1; 3) apoE-mediated ABCA1-dependent cholesterol efflux was not affected by apoE isoforms in fibroblasts; and 4) the lipid translocase activity of ABCA1 generates apoE-containing high density-sized lipoprotein particles. Thus, ABCA1 is essential for the biogenesis of high density-sized lipoprotein containing only apoE particles in vivo.