Plasma triglyceride-rich lipoproteins vary in their lipid composition during metabolism. We investigated the effects of cholesterol (Chol) on the surface properties of lipid emulsions and on the interactions with two amphipathic peptides, acetyl-DWLKAFYDKVAEKLKEAF-amide (Ac-18A-NH(2)) and acetyl-KWLDAFYDEVAEKLICKAF-amide (Ac-18G*NH(2)), which differ in charge distribution. The fluorescence lifetimes of N-dansyl phosphatidylethanolamine (dansyl-PE) and n-(9-anthroyloxy)stearic acid (n-AS, n = 2, 6, and 12) were used to assess the water penetration into the headgroup and acyl chain regions of phosphatidylcholine (PC), respectively. Steady-state fluorescence anisotropy of n-AS was also performed to evaluate the acyl chain fluidity in emulsion surface monolayers. Chol decreased the fluorescence lifetime of dansyl-PE and increased the lifetimes and anisotropy values of n-AS. These results demonstrated that Chol alters the surface properties of emulsions, i.e., induces PC headgroup separation and acyl chain condensation. The two peptides showed different responses to Chol in several experiments: Addition of Chol to emulsions decreased and increased the dissociation constants of Ac-18A-NH(2) and Ac-18G*-NH(2), respectively. Furthermore, the a-helical content of Ac-18A-NH(2) was decreased by Chol, whereas that of Ac-18G*-NH(2) was unchanged. The higher reduction in helicity for Ac-18A-NH(2) is probably due to its deeper penetration than Ac-18G*-NH(2) into the hydrocarbon region of surface monolayers in the absence of Chol, which was demonstrated by Trp quenching experiments with n-AS. From these results, the charge distribution of the amphipathic helices is suggested to be a determining factor in their response to Chol enrichment in emulsions.