Infusion of PEGylated liposomes can give rise to hypersensitivity reactions (HSRs) in a relatively large number of patients. Previously it has been shown that these reactions can be caused by activation of the complement (C) system by a negative charge on the anchor molecule of PEG at the liposomal surface. In this study it is tested whether the activation of the C system by PEG-liposomes could be significantly reduced to values comparable to nonreactive liposomal formulations, by changing the PEGylation-profile on the liposomal surface. Therefore, the formation of C activation markers SC5b-9, C3a, C4d and Bb in normal human serum by both prednisolone loaded and empty liposomes with a variation of PEG chain length, PEG surface concentration, PEG anchor molecule and liposomal size was determined using in vitro assays. The tested liposomes caused no or only mild (30%) activation of C except for one formulation wherein the PEG2000 was anchored to cholesterol (CHOL-PEG2000). The latter liposomes caused paralleling rises in SC5b-9 and Bb levels, suggesting excess activation of the alternative pathway. While the relative safety of weak C activator liposomes remains to be confirmed in vivo, the unique, non-charge and non-antibody-mediated direct conversion of C3 by CHOL-PEG2000 liposomes (although argues against the clinical development of these vesicles) opens new opportunities to understand liposomal C activation at the molecular level.