The mechanism of pH-induced destabilization of liposomes composed of phosphatidylethanolamine and a charged cholesteryl ester was studied by following the release of encapsulated aqueous contents. The kinetics of release were measured continuously by using the water-soluble fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid in combination with the water-soluble quencher p- xylylenebis (pyridinium) bromide. With this fluorescence assay, release of contents from liposomes composed of phosphatidylethanolamine and cholesteryl hemisuccinate was shown to be a function of pH, ratio of phosphatidylethanolamine to cholesteryl hemisuccinate, and acyl chain composition of the phosphatidylethanolamine. Leakage was very slow at pH 5.5 and increased dramatically with decreasing pH down to 4.0. Replacing phosphatidylethanolamine by phosphatidylcholine eliminated the effect of pH on leakage. Analysis of the kinetics of release by a mass action model demonstrated that bilayer destabilization and leakage occur subsequent to aggregation. The requirement of bilayer contact for destabilization has been found previously for acidic phospholipid bilayers in the presence of divalent cation and for saturated phosphatidylcholine bilayers below the isothermal phase transition temperature. The phosphatidylethanolamine-containing bilayers examined here satisfy the same requirement.