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A novel acid-labile poly(ethylene glycol) (PEG)-conjugated lipid, (R)-1,2-di-O-(1′Z,9′Z-octadecadienyl)glyceryl-3-(ω-methoxy-poly(ethylene glycolate, MW5000) (BVEP), a neutral PEG-derivatized analogue of diplasmenylcholine, has been used at low molar ratios to disperse the nonlamellar, fusogenic lipid 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as unilamellar liposomes. It was anticipated that acidcatalyzed hydrolysis of the vinyl ether linkages would destabilize BVEP/DOPE liposomes by removal of the water-soluble, sterically stabilizing PEG layer, thereby promoting contents release and membranemembrane fusion. This paper describes the hydrolysis rates, contents release rates, and fusion kinetics of BVEP-stabilized DOPE liposomes at 1:99, 3:97, and 5:95 molar ratios of BVEP/DOPE. Calcein leakage kinetics indicate that 3:97 BVEP/DOPE liposomes offer the best stability at pH 7.4 while retaining favorable leakage properties at pH 4.5 (t50%release ≈ 4 h). N-Rhodamine phosphatidylethanolamine/N-nitrobenzoxadiazole phosphatidylethanolamine lipid mixing assays show that membrane fusion occurs on a much slower time scale than leakage in these systems, with ∼12% lipid mixing occurring over a 24 h time period at pH 2.0. No appreciable membrane fusion occurred in these liposomes at either pH 7.4 or 4.5 when monitored for up to 3 days. 31 P NMR spectra at pH 7.4 contain a single isotropic line shape, consistent with the presence of large liposomes. The 31 P NMR line shape did not change significantly even after long exposure times at pH 4.0; however, Mn 2+ addition experiments with acid-treated samples produced linebroadened spectra, indicating that all the phosphorus sites were continuous with the bulk water phase. Time-dependent cryogenic transmission electron microscopy experiments indicate that extensive liposome collapse to give small dense aggregates occurs over a 1-4 h period when 3:97 BVEP/DOPE liposomes are acidified to pH 4.5. Taken together, these results suggest that acid-catalyzed hydrolysis of BVEP/DOPE liposomes does result in dePEGylative triggering; however, the primary outcome of this cleavage process is contents leakage and liposome collapse to give <100 nm particles that are presumed to be inverted hexagonal phase structures, with membrane lipid mixing occurring on a kinetically slower time scale. † Dedicated to the memory of David F. O'Brien, deceased July 2002.