We investigated the interaction of liposomes of different surface properties with two mammalian cell lines, CV1, an African green monkey kidney cell line, and J774, a murine macrophage-like cell line. Cell surface binding and endocytosis of liposomes were quantified by fluorometry, using the liposome-encapsulated pH-sensitive fluorescent dye, pyranine, and the lipid marker rhodamine-PE. The liposome uptake was dependent both on the surface properties of the liposomes and on the cell line. Negatively charged phospholipids incorporated into egg phosphatidylcholine (PC)/cholesterol (C) (2:1) liposomes were recognized by the two cell lines to different extents depending on the lipid headgroup and its charge density in the liposome bilayer. Inclusion of 9% phosphatidylserine (PS), phosphatidylglycerol (PG), or phosphatidic acid (PA) promoted the uptake by CV1 cells more than 20-fold. Increasing the content of these negatively charged lipids beyond 9% did not further enhance the uptake. In contrast, 9% monosialoganglioside GM1, phosphatidylinositol (PI), or phosphatidylethanolamine conjugated to poly(ethylene glycol) (PEG-PE) did not promote the uptake. Inclusion of 9% PS, PG, PA or PI in PC/C liposomes did not enhance the uptake by J774 cells, but a drastic enhancement was observed when increasing concentrations of these anionic lipids were incorporated in the liposome bilayer. At least 50% PS, PG, or PI was needed to reach the level of uptake seen with CV1 cells. The uptake of liposomes containing 50% PS by J774 cells was inhibited by poly-anions which are the competing ligands for scavenger receptors, but the uptake by CV1 was not inhibited. Different mechanisms of liposome uptake by these two cell lines are suggested from the different patterns of uptake and the competition with various poly-anions. The differences observed in the uptake rate of liposomes with different lipid compositions seemed to be primarily due to the differences in the binding between liposomes and cell membrane components. The in vitro interaction of various liposomes with these cell lines, especially CV1 cells, shows significant similarities to the in vivo clearance rates of the liposomes.