We recently reported that a second dose of polyethylene glycol (PEG) (M.W. 2000)-modified liposomes (mPEG2000-liposomes) is rapidly cleared from the blood and accumulates in the liver when injected twice in the same rat or mouse at several-day intervals (referred to as the "accelerated blood clearance (ABC) phenomenon"). In the present study we observed that a high dose (5 micromol/kg) of conventional liposomes (CL: without a PEG-coating) can induce the same phenomenon, while a low lipid dose (0.001 micromol/kg) did not. The induction of the phenomenon by mPEG2000-liposomes decreased with increasing first dose (0.001-5 micromol/kg). We observed a strong inverse relationship between the dose of initially injected PEG2000-liposomes and the extent to which the ABC phenomenon was induced: the higher the dose the smaller the phenomenon. Increasing the PEG density at the liposome surface beyond 5 mol% attenuated rather than induced the induction of the phenomenon, but elongation of the PEG chain length up to M.W. 5000, had no effect. In a series of hematological, serum-biochemical and histopathological safety evaluations we observed neither acute toxicity nor any signs of hepatic damage during the induction of the ABC phenomenon. Morphological examination of the liver by transmission electron microscopy (TEM) showed extensive accumulation of the second dose of mPEG2000-liposomes in the Kupffer cells, even already after 15 min, suggesting that the PEG liposomes had somehow lost the protective effect of the surface-grafted PEG against rapid clearance. The observations reported in this paper may have a considerable impact on the design and engineering of PEGylated liposomal formulations for use in multiple drug therapy.