By use of a carboxylated derivative of phosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine (COPE), pH-sensitive liposomes have been designed that have a wide range of leakage properties. The leakage rate of the vesicle contents, as determined by the release kinetics of the water-soluble fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid complexed with the quencher p-xylenebis(pyridinium) dibromide [Ellens, H., Bentz, J., & Szoka, F. C. (1984) Biochemistry 23, 1532], was found to be dependent on the lipid composition and the pH of the incubation medium. Pure COPE vesicles released their encapsulated contents at pH 7.4 but not at pH 4.0. Leakage of these vesicles appears to be due to the electrostatic interactions between the COPE molecules. A dramatic reversal of the leakage properties was observed in mixed-lipid vesicles composed of COPE containing increasing amounts of dioleoylphosphatidylethanolamine (DOPE). Unlike pure COPE vesicles, COPE/DOPE (3:7) vesicles were more leaky under acidic conditions (pH 4.0) than they were at neutral pH. Studies employing a fluorescent COPE analogue, N-succinyl-1-acyl-2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]ca proyl]phosphatidylethanolamine, suggested that the mechanism of leakage might be a result of lipid-packing defects due to the nonbilayer properties of DOPE and protonation of the COPE molecules. Hence, the mechanism of release is different from that of other recently described pH-sensitive liposomes where either fusion or aggregation of the vesicles results in the release of vesicle contents.