The cytoplasmic domain of B ephrins plays a central role in bidirectional signal transduction processes controlling pattern formation and morphogenesis, such as axon guidance, cell migration, segmentation, and angiogensis. In particular, the extremely conserved last 33-residue cytoplasmic subdomain was shown to bind to both a PDZ domain for one signaling pathway [Lu et al. (2001) Cell 105, 69-79] and an SH2 domain from an alternative signaling network [Cowan and Henkemeyer (2001) Nature 413, 174-179]. To date, no structural information is available for the cytoplasmic domain of ephrin B proteins. We report here a detailed NMR study on the structural and dynamic properties of the cytoplasmic domain of human ephrin B2. Our results reveal the following: (1) the N-terminal region of the cytoplasmic domain from residues 253 to 300 lacks the ability for structure formation and is particularly prone to aggregation; and (2) the C-terminal functional subdomain from residues 301 to 333 assumes two distinctive structural elements with residues 301-322 adopting a well-packed hairpin structure followed by a flexible C-terminal tail. Furthermore, the backbone (15)N relaxation data demonstrate that the hairpin structure has significantly limited backbone motions, indicating a high conformational stability for the folded structure. Therefore, while the flexible C-terminal tail is suitable for binding to the PDZ domain, the folded hairpin may represent a latent structure requiring phosphorylation-induced conformational changes for high-affinity interactions with the SH2 domain.