The first step in removing cholesterol from a cell is the ATP-binding cassette transporter 1 (ABCA1)-driven transfer of cholesterol to lipid-free or lipid-poor apolipoprotein A-I (apoA-I), which yields cholesterol-rich nascent high-density lipoprotein (nHDL) that then matures in plasma to spherical, cholesteryl ester-rich HDL. However, lipid-free apoA-I has a three-dimensional (3D) conformation that is significantly different from that of lipidated apoA-I on nHDL. By comparing the lipid-free apoA-I 3D conformation of apoA-I to that of 9–14 nm diameter nHDL, we formulated the hypothetical helical domain transitions that might drive particle formation. To test the hypothesis, ten apoA-I mutants were prepared that contained two strategically placed cysteines several of which could form intramolecular disulfide bonds and others that could not form these bonds. Mass spectrometry was used to identify amino acid sequence and intramolecular disulfide bond formation. Recombinant HDL (rHDL) formation was assessed with this group of apoA-I mutants. ABCA1-driven nHDL formation was measured in four mutants and wild-type apoA-I. The mutants contained cysteine substitutions in one of three regions: the N-terminus, amino acids 34 and 55 (E34C to S55C), central domain amino acids 104 and 162 (F104C to H162C), and the C-terminus, amino acids 200 and 233 (L200C to L233C). Mutants were studied in the locked form, with an intramolecular disulfide bond present, or unlocked form, with the cysteine thiol blocked by alkylation. Only small amounts of rHDL or nHDL were formed upon locking the central domain. We conclude that both the N- and C-terminal ends assist in the initial steps in lipid acquisition, but that opening of the central domain was essential for particle formation.