For peripheral iron to reach the brain, it must transverse the blood-brain barrier. In order for the brain to obtain iron, transferrin receptors are present in the vascular endothelial cell to facilitate movement of transferrin bound iron into the brain parenchyma. However, a number of significant voids exist in our knowledge about transport of iron into the brain. These gaps in our knowledge are significant not only because iron is an essential neurotrophic factor but also because the system for delivery of iron into the brain is being viewed as an opportunity to circumvent the blood-brain barrier for delivery of neurotoxins to tumors or trophic factors in neurodegenerative diseases. In this study, we have used fluorescein-transferrin-59Fe in a bovine retinal endothelial cell culture system to determine the mechanism of transferrin-iron transport and to test the hypothesis that the iron status of the endothelial cells would influence iron transport. Our results indicated that iron is transported across endothelial cells both bound to and not bound to transferrin. The ratio of non-transferrin-bound iron to transferrin-bound iron transported is dependent upon the iron status of the cells. Blocking acidification of endosomes led to a significant decrease in transport of non-transferrin-bound iron but not transferrin-bound iron. Blocking pinocytosis had no effect on either transferrin or iron transcytosis. These results indicate that there is both transferrin-mediated and non-transferrin-mediated transcytosis of iron and that the process is influenced by the iron status of the cells. These data have considerable implications for common neurodegenerative diseases that are associated with excess brain iron accumulation and the numerous neurological complications associated with brain iron deficiency.