The in vitro uptake of phospholipid vesicles by mouse leukemia L1210 cells was examined. Liposomes were generated by prolonged ultrasonic dispersion of aqueous dispersions of mixed lipids in the presence of radiolabeled inulin. Multilamellar vesicles were separated from unilamellar vesicles by column chromatography. Vesicle populations were examined by electron microscopy. Neutral vesicles were generated from egg yolk phosphatidylcholine and cholesterol, and surface charge was introduced via either phosphatidylserine or octadecylamine. Uptake, measured as cell-associated radioactivity, was temperature dependent and was strongly decreased by metabolic inhibitors. These results suggested that liposomes are taken up to a major extent by an energy-dependent mechanism. The uptake of liposomes by cells of a young culture was about 2-fold higher than was the uptake of liposomes by cells of a stationary culture. The uptake of positively charged liposomes by cells was about 20-fold higher than that of either neutral or negatively charged vesicles. About one-half of the cell-associated radioactivity transferred by positively charged liposomes could be removed by cell surface treatment with trypsin or neuraminidase or by a short exposure to 0.6 N NaCl.