Between several experiments, we recorded similar values of uptake followed invariably by equivalent rates of transcytosis and recycling of the internalized ligand. High values of transcytosed/recycled ligand from our assay can be explained by the level of membrane-localized TfR on hCMEC/D3 cells compared to primary human brain endothelial cells. At similar culture confluency, the immortalized cells consistently show higher levels of membrane-resident TfR than primary human endothelial cells. The work of Raub and Newton and Descamps et al. have described the paucity of membrane-localized TfR in confluent cultures of primary endothelial cells and hence the published values of transcytosis from primary cell cultures are generally low. The ratio of transferrin transcytosis to recycling was described as 1:3 in primary bovine brain endothelial cells ; by contrast, we observe similar rates for both processes. It cannot be excluded that this difference, in line with the higher TfR expression level, could also be due to slightly distorted sorting of the transferrin receptor in the immortalized cell line; alternatively, we cannot rule out the possibility that some material unspecifically bound to the cells and is only released after prolongued incubation. Following our validation of the assay protocol with the ligand, we proceeded to test antibodies against putative transcytosis receptors for transport. Targeting of receptors, particularly the insulin and transferrin receptors by chimeric peptides and antibodies has been suggested to be an effective way of delivering drugs to the brain in several animal models. The OX-26 murine monoclonal antibody to the rat TfR and the 83–14 murine monoclonal antibody to the human insulin receptor are the best known examples of antibodies with published BBB permeability properties. We tested an antibody to the IGF-1R, described for its capacity to engage in transcytotic activity. Our results indicate slight intracellular degradation and significant recycling of the antibody to the apical surface, but no transcytosis. Investigation of IGF-1R mediated transcytosis has implicated facilitation of the process by the association with LRP1 in the rat brain. We could not detect membrane-resident LRP1 in hCMEC/D3 cells. Reports showing Lrp1 expression in hCMEC/D3 were obtained using methods which detect both extracellular and intracellular protein. Absence of membrane LRP1 could offer an explanation for the lack of transcytosis of an IGF-1R mAb in our model system. We generated the 128.1 anti-TfR antibody described in Friden et al., because it had been suggested to access the brain in Cynomolgous monkeys after intravenous application. In hCMEC/D3 cells, the antibody initially appeared to follow the classical uptake and internalization through clathrin-coated vesicles and subsequent localization in early endosomes.