Earlier we reported that PEGylated liposomes lose their long-circulating characteristic when they are administrated twice in the same animal with certain intervals (referred to as the accelerated blood clearance (ABC) phenomenon). We proposed that anti-PEG IgM, induced by the PEGylated liposomes, is responsible for the phenomenon, based on the observation that IgM thus produced selectively binds to the surface of PEGylated liposomes, subsequently leading to substantial complement activation. Interestingly, we found that under certain circumstances administration of conventional liposomes without PEG-coating also caused a strong ABC response upon injection of a second dose of PEGylated liposomes, but not of conventional liposomes. This suggests that also conventional liposomes not modified with PEG can promote an IgM response against PEG. We report here that, irrespective of the presence or absence PEG-coating, a single first dose of liposomes is capable of inducing a strong anti-PEG IgM response and, under certain circumstances, also weak responses against other lipid components. A good correspondence was observed between the amount of IgM associating with both PEGylated and conventional liposomes, concomitant complement activation triggered by those liposomes and the magnitude of the ABC phenomenon against those liposomes. Taken together, our results demonstrate that the ABC phenomenon is fully attributable to production of anti-PEG IgM by the first dose of liposomes and the subsequent complement activation upon a second dose of PEGylated but not conventional liposomes. Although the responsible immunogenic epitopes of the liposomes remain to be determined, the immunogenicity of 'empty' liposomes presents a serious concern in the development of liposomal formulations and their use in the clinic. Furthermore, our findings as described here raise important concerns with regard to the safety and efficiency of liposomes currently under development for clinical use.