The oligomeric structure of ABCA1 transporter and its function related to the biogenesis of nascent apoA-I-containing particles (LpA-I) were investigated. Using n-dodecylmaltoside and perfluoro-octanoic acid combined with non-denaturing gel, the majority of ABCA1 was found as a tetramer in ABCA1-induced human fibroblasts. Furthermore, using chemical cross-linking and SDS-PAGE, ABCA1 dimers but not the tetramers were found covalently linked. Oligomeric ABCA1 was present in isolated plasma membranes as well as in intracellular compartments. Interestingly, apoA-I was found to be associated with both dimeric and tetrameric, but not monomeric, forms of ABCA1. Neither apoA-I nor lipid molecules did affect ABCA1 oligomerization. Immunoprecipitation analysis showed that oligomeric ABCA1 did not contain other associated proteins. We next investigated the relationship between the oligomeric ABCA1 complex and the structure of LpA-I. Lipid-free apoA-I incubated with normal cells generated LpA-I with diameters between 9.5 and 20 nm. Subsequent isolation of LpA-I followed by cross-linking revealed the presence of four and eight apoA-I molecules per particle, whereas apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles and remained in the monomeric form. These results demonstrate that: 1) ABCA1 exists as an oligomeric complex; and 2) ABCA1 oligomerization was independent of apoA-I binding and lipid molecules. The findings that the majority of ABCA1 exists as a tetramer that binds apoA-I, together with the observation that LpA-I contains at least four molecules of apoA-I per particle, support the concept that the homotetrameric ABCA1 complex constitutes the minimum functional unit required for the biogenesis of high density lipoprotein particles.