Rapid internalization of drugs from delivery vehicles via non-endocytotic pathways is an important goal. The transport of imaging probes attached to cholesterol and introduced via a liposomal formulation is considered here, in order to evaluate the intracellular distribution and kinetics of small molecular cargo that might be attached to cholesterol or phospholipids. The internalization efficiencies of two fluorescent cholesterol analogues, one carrying a fluorophore on the head of the cholesterol molecule 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate (BODIPY)-cholesteryl ester (CE) (BODIPY-CE) and the other on the tail (25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl]amino]-27-norcholesterol (NBD-cholesterol)), were compared with those of other phospholipid molecules (NBD-phosphatidylcholine (PC) and NBD-phosphatidylethanolamine (PE)) using a liposomal formulation (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 85.5 M%; 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2k), 9.5 M%; fluorescent analogue, 5 M%). The rate and transfer efficiency were NBD-cholesterol>BODIPY-CE>NBD-PC>NBD-PE. NBD-cholesterol, delivered by liposomes with an average diameter of 100 nm, localized in the perinuclear region and lipid storage droplets, with transfer observed in as little as 5 min. NBD-cholesterol transport was approximately constant with time, suggesting a unidirectional mode of entry. In the absence of PEG within the liposome, the transfer rate decreased. Filipin, a caveolae-blocking agent, caused 70% inhibition of cholesterol internalization in treated cells, suggesting that cholesterol internalization follows a caveolae-mediated pathway.