We have recently reported that PEGylated liposomes (PL) are cleared rapidly from the blood circulation when they are administered twice in the same rat at certain intervals, even if the liposomes are sterically stabilized by a surface modification with PEG (referred to as the accelerated blood clearance (ABC) phenomenon, J. Control. Release, 88, 35-42 (2003)). Now we report on the influence of physicochemical properties (PEG-modification, size and surface charge) of either the first or the second dose of liposomes on the ABC phenomenon. When, for the first dose, conventional liposomes (CL; without a PEG coating) of 110-nm diameter were injected, only a very slight ABC phenomenon was observed, irrespective of the liposomal surface charge: both clearance rate and hepatic accumulation of the second injected PL were only slightly enhanced compared to those of a single dose of PL. Interestingly, when for the first injection small-size liposomes (60 nm) were used, either charged or PEG-modified, but not neutral, the ABC phenomenon was clearly manifest. Apparently, the induction of the ABC phenomenon is not only determined by the PEG coating but also by the size and surface charge of the first dose of liposomes. Also when for the second dose small-size PEGylated liposomes were used, the ABC phenomenon was observed after induction by a first injection of PL, whereas plasma kinetics and organ uptake of a second dose of negatively charged CL (NCL, 110 nm) or small-sized NCL (SNCL, 60 nm) were not altered. Apparently, the PEG coating on the second dose is essential for the liposomes to be susceptible to the ABC phenomenon. The results reported here suggest that the physicochemical properties of both the first and second dose of liposomes are important either for the induction of the phenomenon or for its expression. Our observations may have a considerable impact on the clinical application and engineering of liposomal formulations for use in multiple drug therapy.