The acid-labile poly(ethyleneglycol)-diorthoester-distearoylglycerol lipid (POD), was used with a cationic lipid-phosphatidylethanolamine mixture to prepare stabilized plasmid-lipid nanoparticles (POD SPLP) that could mediate gene transfer in vitro by a pH triggered escape from the endosome. Nanoparticles of 60 nm diameter were prepared at pH 8.5 using a detergent dialysis method. The DNA encapsulation efficiency in the nanoparticles was optimal between 10 and 13 mol % ratio of cationic lipid and at a POD content of 20 mol %. The apparent zeta potential of the nanoparticles at 1 mM salt and pH 7.5 was positive, indicating cationic lipid on the external surface. However, the external layer of the nanoparticles was depleted in the cationic component compared to the starting mole ratio. Low pH sensitivity of the POD SPLP was characterized by a lag phase followed by a rapid collapse; at pH 5.3 the nanoparticles collapsed in 100 min. Nanoparticles prepared from a pH-insensitive PEG-lipid, PEG-distearoylglycerol had similar physicochemical characteristics as the POD SPLP but did not collapse at low pH. The POD SPLP had up to 3 orders of magnitude greater gene transfer activity than did the pH-insensitive nanoparticles. Both the pH-sensitive and pH-insensitive nanoparticles were internalized to a qualitatively similar extent in a punctate pattern into cultured cells within 2 h of incubation with the cells; thus, increased gene transfer of the POD SPLP was due to a more rapid escape from the endosome rather than to greater cell association of these nanoparticles. These results suggest that the pH-sensitive stabilized plasmid-lipid nanoparticles may be a useful component of a synthetic vector for parenterally administered gene therapy.