To test the hypothesis that sidedness of interfacial arginine (Arg) in apoA-I mimetic peptides, similar to that observed in apoA-I (Bashtovyy, D. et al. 2011. Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function. J. Lipid Res. 52: 435-450.), may be important for biological activity, we compared properties of 4F and analogs, [K 4,15>R]4F and [K 9,13>R]4F, with Lys>Arg substitutions on the right and left side, respectively, of the 4F amphipathic helix. Intraperitoneal administration of these peptides into female apoE null mice (n = 13 in each group) reduced en face lesions significantly compared with controls; 4F and [K 4,15>R]4F were equally effective whereas [K 9,13>R]4F was less effective. Turnover experiments indicated that [K 4,15>R]4F reached the highest, whereas [K 9,13>R]4F had the lowest, plasma peak levels with a similar half life as the [K 4,15>R]4F analog. The half life of 4F was two times longer than the other two peptides. The order in their abilities to associate with HDL in human plasma, generation of apoA-I particles with pre-β mobility from isolated HDL, lipid associating ability, and sensitivity of lipid complexes to trypsin digestion was: 4F>[K 4,15,>R]4F>[K 9,13>R]4F. These studies support our hypothesis that the sidedness of interfacial Arg residues in the polar face of apoA-I mimetics results in differential biological properties. Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.