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.