Eph receptors and their membrane-associated ephrin ligands mediate cell-cell repulsion to guide migrating cells and axons. Repulsion requires that the ligand-receptor complex be removed from the cell surface, for example by proteolytic processing of the ephrin ectodomain. Here we show that cell contact-induced EphB-ephrinB complexes are rapidly endocytosed during the retraction of cells and neuronal growth cones. Endocytosis occurs in a bi-directional manner that comprises of full-length receptor and ligand complexes. Endocytosis is sufficient to promote cell detachment and seems necessary for axon withdrawal during growth cone collapse. Here, we show a mechanism for the termination of adhesion and the promotion of cell repulsion after intercellular (trans) interaction between two transmembrane proteins.