Human apolipoprotein C-I (apoC-I) is an exchangeable apolipoprotein that binds to lipoprotein particles in vivo. In this study, we employed a LC-MS/MS assay to demonstrate that residues 38-51 of apoC-I are significantly protected from proteolysis in the presence of 1,2-dimyristoyl-3-sn-glycero-phosphocholine (DMPC). This suggests that the key lipid-binding determinants of apoC-I are located in the C-terminal region, which includes F42 and F46. To test this, we generated site-directed mutants substituting F42 and F46 for glycine or alanine. In contrast to wild-type apoC-I (WT), which binds DMPC vesicles with an apparent Kd [Kd(app)] of 0.89 microM, apoC-I(F42A) and apoC-I(F46A) possess 2-fold weaker affinities for DMPC with Kd(app) of 1.52 microM and 1.58 microM, respectively. However, apoC-I(F46G), apoC-I(F42A/F46A), apoC-I(F42G), and apoC-I(F42G/F46G) bind significantly weaker to DMPC with Kd(app) of 2.24 microM, 3.07 microM, 4.24 microM, and 10.1 microM, respectively. Sedimentation velocity studies subsequently show that the protein/DMPC complexes formed by these apoC-I mutants sediment at 6.5S, 6.7S, 6.5S, and 8.0S, respectively. This is compared with 5.0S for WT apoC-I, suggesting the shape of the particles was different. Transmission electron microscopy confirmed this assertion, demonstrating that WT forms discoidal complexes with a length-to-width ratio of 2.57, compared with 1.92, 2.01, 2.16, and 1.75 for apoC-I(F42G), apoC-I(F46G), apoC-I(F42A/F46A), and apoC-I(F42G/F46G), respectively. Our study demonstrates that the C-terminal amphipathic alpha-helix of human apoC-I contains the major lipid-binding determinants, including important aromatic residues F42 and F46, which we show play a critical role in stabilizing the structure of apoC-I, mediating phospholipid interactions, and promoting discoidal particle morphology.