In this study, we investigated the contribution of the complement system to the biodistribution of phosphatidylserine (PS)-containing liposomes in rat and guinea pig. It appeared that the inclusion of PS in the liposome formulation accelerates the rate of liposome uptake by liver, resulting in rapid elimination of the liposomes from blood circulation. Pretreatment with K76COOH (K76), an anti-complement agent, decreased the rapid uptake of PS-containing liposomes by guinea pig liver, resulting in increasing blood concentration of the liposomes. Significant complement-dependent liposome destabilization was observed in vitro in both animals, whereas the complement-dependent destabilization in vivo was likely only a part of the process of the clearance of the PS-containing liposomes. This discrepancy suggests that the rate of complement-dependent liposome uptake by liver is much faster than the rate of complement-dependent liposome destabilization in vivo. Pretreatment of K76 dramatically inhibited the binding of C3 fragments, one of dominant opsonins, to PS-containing liposomes in guinea pig under both in vivo and in vitro conditions. This finding suggests that the C3 fragments in the system are responsible for the clearance of the PS-containing liposomes in guinea pig. In rat, in contrast to guinea pig, in vivo binding of C3 fragments was not inhibited by K76-pretreatment, while in vitro binding was inhibited. This discrepancy may be due to different experimental conditions between in vitro and in vivo assay. Nevertheless, based on the observations in this study, the complement components are most likely involved in the clearance of the PS-containing liposomes in rat. Taken together, the activity of PS in enhancing the liposome clearance appears to be mediated by the complement components, presumably C3 fragments, in both guinea pig and rat. This is a first report showing the mechanism on the hepatic uptake of the PS-containing liposomes in guinea pig.