In mature human apolipoprotein A-I (apo A-I), the amino acid residues 1- 43 are encoded by exon 3, whereas residues 44-243 are encoded by exon 4 of the apo A-I gene. The region encoded by exon 4 of the apo A-I gene contains 10 tandem amphipathic α-helixes; their location and the class to which they belong are as follows: helix 1 (44-65, class A1), helix 2 (66-87, class A1), helix 3 (88-98, class Y), helix 4 (99-120, class Y), helix 5 (121-142, class A1), helix 6 (143-164, class A1), helix 7 (165-186, class A1), helix 8 (187-208, class A1), helix 9 (209-219, class Y), and helix 10 (220-241, class Y). To examine the effects of multiple tandem amphipathic helixes compared to individual helixes of apo A-I on lipid association, we have studied lipid-associating properties of the following peptides: Ac-44-87-NH2 (peptide 1-2), Ac-66-98-NH2 (peptide 2-3), Ac-66-120-NH2 (peptide 2-3-4), Ac88-120-NH2 (peptide 3-4), Ac-99-142-NH2 (peptide 4-5), Ac-121-164-NH2 (peptide 5-6), Ac-143-186-NH2 (peptide 6-7), Ac-165-208-NH2 (peptide 7-8), Ac-187-219-NH2 (peptide 8-9), and Ac-209-241-NH2 (peptide 9-10). To study lipid-associating properties of the region encoded by exon 3 of the apo A-I gene, 1-33-NH2 (peptide G*) has also been studied. The results of the present study indicate that, among the peptides studied, peptides 1-2 and 9- 10 possess significantly higher lipid affinity than the other peptides, with peptide 9-10 having higher lipid affinity than peptide 1-2, as evidenced by (i) higher helical content in the presence of 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), (ii) faster rate of association with DMPC multilamellar vesicles (MLV), (iii) greater reduction in the enthalpy of gel to liquid, crystalline phase transition of DMPC MLV, (iv) higher exclusion pressure from an egg yolk phosphatidylcholine monolayer, and (v) higher partitioning into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine MLV. A comparison of the free energies of lipid association (ΔG) of the peptides studied here with those studied previously by us [Palgunachari, M. N., et al. (1996) Arterioscler. Thromb. Vasc. Biol. 16, 328-338] indicates that, except for the peptides 4-5 and 5-6, other peptides possess higher lipid affinities compared to constituent helixes. However, the lipid affinities of the peptides studied here are neither higher than nor equal to the sum of the lipid affinities of the constituent helixes. This indicates the absence of cooperativity among the adjacent amphipathic helical domains of apo A-I for lipid association. As indicated by ΔG, the lipid affinity of peptide 4-5 is higher than peptide 5 but lower than peptide 4; the lipid affinity of peptide 5-6 is lower than both peptides 5 and 6. Implications of these results for the structure and function of apo A-I are discussed.