Two homologous apoA-I mimetic peptides, 3F-2 and 3F14, differ in their in vitro antiatherogenic properties (Epand, R. M., Epand, R. F., Sayer, B. G., Datta, G., Chaddha, M., and Anantharamaiah, G. M. (2004) J. Biol. Chem. 279, 51404-51414). In the present work, we demonstrate that the peptide 3F-2, which has more potent anti-inflammatory activity in vitro when administered intraperitoneally to female apoE null mice (20 μg/mouse/day) for 6 weeks, inhibits atherosclerosis (lesion area 15,800 ± 1000 μm2, n = 29), whereas 3F14 does not (lesion area 20,400 ± 1000 μm2, n = 26) compared with control saline administered (19,900 ± 1400 μm2, n = 22). Plasma distribution of the peptides differs in that 3F-2 preferentially associates with high density lipoprotein, whereas 3F14 preferentially associates with apoB-containing particles. After intraperitoneal injection of 14C-labeled peptides, 3F14 reaches a higher maximal concentration and has a longer half-time of elimination than 3F-2. A study of the effect of these peptides on the motional and organizational properties of phospholipid bilayers, using several NMR methods, demonstrates that the two peptides insert to different extents into membranes. 3F-2 with aromatic residues at the center of the nonpolar face partitions closer to the phospholipid head group compared with 3F14. In contrast, only 3F14 affects the terminal methyl group of the acyl chain, decreasing the 2H order parameter and at the same time also decreasing the molecular motion of this methyl group. This dual effect of 3F14 can be explained in terms of the cross-sectional shape of the amphipathic helix. These results support the proposal that the molecular basis for the difference in the biological activities of the two peptides lies with their different interactions with membranes. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.