The surface potentials and the packing properties of phospholipid liposomes containing anionic lipids with covalently attached poly(ethylene glycol) polymers (PEG(2000)-PE) were investigated. The negative surface potential induced by the PEG-lipid in EPC/cholesterol and SPC/cholesterol liposomes was determined by means of titration of the fluorescent probe 4-heptadecyl-7-hydroxycoumarin (HC). The shift in the apparent pK of the probe corresponds to an induced potential of -13 mV for 5 mol % PEG-lipid in the phospholipid/cholesterol (ionic strength = 150 mM). For EPC liposomes without cholesterol, 5 mol % PEG-lipid did not cause any detectable shift in the pK of the probe. This indicates that the EPC liposome surface in fact carries a net negative charge. The above results were supported by determinations of the potential. The packing order of the outer and inner parts of the phospholipid/cholesterol bilayer membranes was investigated by fluorescence depolarization anisotropy of the probes HC and 1,6-diphenyl-1,3,5-hexatriene (DPH). The PEG-lipid inclusion caused an increase in the anisotropy of HC, located at the water/hydrocarbon interface. In addition, a small increase in anisotropy was observed for DPH upon PEG-lipid inclusion. Since DPH is situated in the hydrocarbon region, these data together with the HC data indicate that the PEG-lipids impose restrictions on the molecular motion in the outer as well as the inner parts of the hydrocarbon region.