Polyethylene glycol (PEG) is used widely in the pharmaceutical industry to improve the pharmacokinetics and reduce the immunogenicity of therapeutic and diagnostic agents. The incorporation of lipid-conjugated PEG into liposomal drug delivery systems greatly enhances the circulation times of liposomes by providing a protective, steric barrier against interactions with plasma proteins and cells. Here we report that liposome compositions containing PEG-lipid derivatives and encapsulated antisense oligodeoxynucleotide (ODN) or plasmid DNA elicit a strong immune response that results in the rapid blood clearance of subsequent doses in mice. The magnitude of this response is sufficient to induce significant morbidity and, in some instances, mortality. This effect has been observed in several strains of mice and was independent of sequence motifs, such as immunostimulatory CpG motifs. The ODN-to-lipid ratio and ODN dose was also determined to be important, with abrogation of the response occurring at a ratio between 0.04 and 0.08 (w/w). Rapid elimination of liposome-encapsulated ODN from blood depends on the presence of PEG-lipid in the membrane because the use of nonpegylated liposomes or liposomes containing rapidly exchangeable PEG-lipid also abrogated the response. These studies have important implications for the evaluation and therapeutic use of liposomal formulations of nucleic acid, as well as the potential development of liposomal vaccines.