The mechanism by which iron enters the central nervous system from the blood is not well understood. Iron in blood plasma is totally bound to transferrin (Tf), a major plasma glycoprotein. Tf receptors are present on the blood-brain barrier (BBB) endothelium. It is not known whether iron separates from Tf during its passage across the endothelial cells and then enters the brain by another mechanism, or whether the two proteins enter the brain together. We characterize here the morphological pathway for endocytosis of a monomeric horseradish peroxidase-transferrin conjugate by the rat BBB endothelium. Our results indicate that this conjugate binds to Tf receptors on the luminal BBB, is internalized via clathrin-coated vesicles, enters early or sorting endosomes, and, subsequently, late or recycling endosomes near the Golgi apparatus. No evidence is found for Tf transcytosis. It is likely that iron separates from Tf in early endosomes, which are assumed to be acidic, as they are in other cells, and enters the brain by an as yet undefined pathway. A clonal line of brain capillary endothelial cells that mimics the BBB when grown on permeabilized membranes can transcytose iron provided as Fe55-Tf. This cell line may provide a useful system to determine the pathway that iron uses to enter the brain. We also present evidence that cultured chick embryo forebrain neurons contain a large number of a unique Tf receptor.