The targeting potential of OX-26-decorated immunoliposomes was investigated, using the human brain endothelial cell line hCMEC/D3 as a model of the blood-brain barrier (BBB). Immuno-nanoliposomes were prepared by the biotin/streptavidin ligation strategy, and their uptake by hCMEC/D3 cells and permeability through cell monolayers was studied. In order to elucidate the mechanisms of uptake, pH-sensitive fluorescence signal of HPTS was used, while transport was measured using double labeled immunoliposomes (with aqueous and lipid membrane fluorescent tags). PEGylated and non-specific-IgG-decorated liposomes were studied under identical conditions, as controls. CHO-K1 cells (which do not overexpress the transferrin receptor) were studied in some cases for comparative purposes. Experimental results reveal that hCMEC/D3 cells are good models for in vitro screening of BBB-targeting nanoparticulate drug delivery systems. Uptake and transcytosis of immunoliposome-associated dyes by cell monolayers was substantially higher compared to those of control liposomes. HPTS-entrapping OX-26-immunoliposome uptake indicated lysosomal localization and receptor-mediated mechanism. The ratio of aqueous/lipid label transport is affected by pre-incubation with antibody, or use of high lipid doses, suggesting that vesicles are transported intact after lysosome saturation. Co-decoration with a second ligand slightly decreases OX-26-decorated vesicle uptake, but not transcytosis, proving that the biotin–streptavidin technique can be applied for the generation of dual-targeting nanoliposomes.