Complement activation causes opsonization of foreign particles leading to particle elimination from the blood. Complement-mediated opsonization of charged and large liposomes presents a fundamental problem in their use to deliver therapeutic agents in vivo. To prolong the circulation half-lives of such liposomes, complement activation must be curtailed. The aim of this study was to assess the ability of poly(ethylene glycol)-lipids (PEG-lipids) to inhibit the in vitro activation of the classical pathway of complement in human serum by anionic liposomes. Incorporation of cholesterol-PEG600 (CH-PEG600), cholesterol-PEG1000 (CH-PEG1000), or phosphatidylethanolamine-PEG2000 (PE-PEG2000) resulted in dose-dependent inhibition of C1q binding and complement activation. The dose of PEG-lipid at which complement activation was blocked was inversely related to the PEG chain length. Complement activation was strongly inhibited when 15 mole% of CH-PEG600, 10 mole% CH-PEG1000, or 5 mole% PE-PEG2000 was incorporated into 100-nm anionic liposomes. PEG-lipid incorporation into larger liposomes (240 nm) was also successful in blocking C1q binding and complement activation. Radiolabeled cholesterol-PEG approximately 1400 was prepared and used to determine both the percentage of CH-PEG incorporated into the liposomes and the percentage maintained in the liposomes in the presence of 50% human serum at 37 degrees C for up to 24 h.