ATI-5261 is a novel, single-helix peptide that stimulates cellular cholesterol efflux with high potency similar to native apolipoproteins on a molar basis. Presently we investigated structural features of the peptide that conferred cholesterol efflux activity. Analogs of ATI-5261 with amino acids arranged in reverse order or with individual arginine (R) to glutamine (Q) substitutions (i.e. R3Q, R14Q or R23Q) stimulated ABCA1 dependent cholesterol efflux similar to ATI-5261. Consequently, neither the presence of specific positively charged residues nor their specific arrangement along the length of the peptide was necessary for mediating cholesterol efflux. Similarly, peptides composed of all D-amino acids stimulated cholesterol efflux efficiently, indicating a stereospecific component was not required for promotion of cholesterol efflux from macrophages. Removal of two or more positively charged residues (R3, 14 -> Q and R3, 14, 23 -> Q) however, greatly reduced the ability of ATI-5261 to mediate cellular cholesterol efflux. This was accompanied by a loss of alpha-helical structure upon dilution, indicating the secondary structure of individual peptide strands was important for stimulating cholesterol efflux. Surprisingly, peptides with removal of two or more positively charged residues retained the ability to bind phospholipid and adopt an alpha-helical structure. These data indicate that the propensity of a hydrophobic peptide to form an amphipathic alpha-helix is not sufficient to mediate cellular cholesterol efflux. Efficient stimulation of cholesterol efflux requires that ATI-5261 retain alpha-helical structure upon dilution. Published by Elsevier Inc.