We have recently demonstrated that anionic liposomes efficiently introduce foreign DNA into postmitotic neurons and other cell types (Lakkaraju, A., Dubinsky, J. M., Low, W. C., and Rahman, Y.-E. (2001) J. Biol. Chem. 276, 32000-32007). To investigate the mechanism of liposome uptake, we followed the internalization of anionic liposome-encapsulated Cy3-1a-beled oligonucleotides (AL-Cy3ONs) by hippocampal neurons using confocal microscopy. Uptake of AL-Cy3ONs was widespread and time- and temperature-dependent, indicative of receptor-mediated endocytosis. The low-density lipoprotein receptor-related protein (LRP) was crucial for anionic liposome endocytosis because the receptor-associated protein or an anti-LRP antibody inhibited internalization, and fibroblasts lacking LRP did not internalize AL-Cy3ONs. Using selective endocytosis inhibitors, we found that liposome endocytosis and intracellular transport required clathrin, dynamin, an intact cytoskeletal network, and phosphatidylinositol 3-kinase activity. Cy3ONs did not significantly colocalize with recycling endosomal/lysosomal markers and entered neuronal nuclei within 1-3 h of incubation. Approximately 50% of the internalized liposomal phospholipids were recycled back to the cell surface, in keeping with the fluidity of their acyl chains. Liposome endocytosis did not require heparan sulfate proteoglycans or cause calcium influx into neurons. Thus, constitutive endocytosis of anionic liposomes by LRP utilizes only one component, in contrast to the more involved heparan sulfate proteoglycan-LRP pathway implicated in the pathogenesis of Alzheimer's disease.