The natural abundance C-13 NMR method was applied to directly determine the binding site of peptides and proteins in lipid bilayers and emulsions on the atomic level. Reliable NMR criteria for the location and depth of peptides and proteins in membranes were shown by the chemical shift and line width analyses, which reproduced not only the deep penetration of a transmembrane channel peptide gramicidin A but also the superficial binding of Ac-18A-NH2 (Ac-DWLKAFYDKVAEKLKEAF-NH2), a synthetic model peptide of amphipathic helices of plasma apolipoprotein A-I (apoA-I). The reliability was ensured by the NMR information, which was consistent with the recent X-ray diffraction study of Ac-18A-NH2 in oriented lipid bilayers (Hristova et al. J. Mol. Biol. 1999, 290, 99). Our method first provided the atomic-level evidence for native apoA-I binding in egg phosphatidylcholine ( EPC) vesicles and triolein (TO)-EPC emulsions as spherical model lipoproteins. Membrane perturbation was most significant at EPC glycerol and ester carbonyl sites when apoA-I was bound to EPC small unilamellar vesicles. This indicates not deep but shallow penetration of apoA-I into the membrane interface whose polarity is intermediate between water and the hydrophobic core. The binding preference for the interfacial site of membranes was confirmed by the common binding site between apoA-I and its model peptide Ac-18A-NH2. Membrane structural modulation by apoA-I was, however, moderate at the bilayer headgroup and the alkyl chain region near the interface. The shallow penetration of apoA-I was also found in TO-EPC emulsions, a protein-free model of triglyceride-rich lipoproteins (chylomicrons) in plasma.