Cubilin, the receptor for intrinsic factor-vitamin B12, is a novel type of high molecular weight receptor consisting of a 27 CUB (complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domain cluster preceded by 8 epidermal growth factor repeats and a short N-terminal sequence. In addition to binding the vitamin B12-carrier complex, cubilin also binds receptor-associated protein. To delineate the structures for membrane association and ligand binding we established a panel of stable transfected Chinese hamster ovary cells expressing overlapping segments of rat cubilin. Analysis of conditioned media and cell extracts of transfected cells revealed that the N-terminal cubilin region conveys membrane association. Helical plotting of this region demonstrated a conserved amphipathic helix pattern (Lys74-Glu109) as a candidate site for hydrophobic interactions. Ligand affinity chromatography and surface plasmon resonance analysis of the secreted cubilin fragments showed ligand binding in the CUB domain region. Further dissection of binding-active fragments localized the binding site for intrinsic factor-vitamin B12 to CUB domains 5-8 and a receptor-associated protein-binding site to CUB domains 13-14. In conclusion, the N-terminal cubilin region seems crucial for membrane association, whereas the CUB domain cluster harbors distinct sites for ligand binding.