The apolipoprotein A-I mimetic peptide 4F (Ac-DWFKAFYDKVAEKEAF-NH 2), with four Phe residues on the nonpolar face of the amphipathic α-helix, is strongly anti-inflammatory, whereas two 3F analogs (3F 3 and 3F14) are not. To understand how changes in helix non-polar face structure affect function, two additional 3F analogs, Ac-DKLKAFYDKVFEWAKEAF-NH2 (3F-1) and Ac-DKWKAVYDKFAEAFKEFL-NH 2 (3F-2), were designed using the same amino acid composition as 3F3 and 3F14. The aromatic residues in 3F-1 and 3F-2 are near the polar-nonpolar interface and at the center of the nonpolar face of the helix, respectively. Like 4F, but in contrast to 3F3 and 3F 14, these peptides effectively inhibited lytic peptide-induced hemolysis, oxidized phospholipid-induced monocyte chemotaxis, and scavenged lipid hydroperoxides from low density lipoprotein. High pressure liquid chromatography retention times and monolayer exclusion pressures indicated that there is no direct correlation of peptide function with lipid affinity. Fluorescence studies suggested that, although the peptides bind phospholipids similarly, the Trp residue in 4F, 3F-1, and 3F-2 is less motionally restricted than in 3F3 and 3F14. Based on these results and molecular modeling studies, we propose that the arrangement of aromatic residues in class A amphipathic helical molecules regulates entry of reactive oxygen species into peptide-phospholipid complexes, thereby reducing the extent of monocyte chemotaxis, an important step in atherosclerosis.