Cellular and molecular mechanisms underlying opioid tolerance and dependence remain elusive. We investigated roles of EphB receptor tyrosine kinases— which play important roles in synaptic connection and plasticity during development and in the matured nervous system—in development and maintenance of physical dependence on morphine in the mouse spinal cord (SC). Spinal administration of an EphB receptor blocking re‐agent EphB2‐Fc prevents and/or suppresses behavioral responses to morphine withdrawal and associated induction of c‐Fos and depletion of calcitonin gene‐related peptide. Western blotting and immunohistochemical flu‐orescence staining demonstrates that EphB1 receptor protein is significantly up‐regulated in the spinal dorsal horn following escalating morphine treatment. Chronic morphine exposure and withdrawal significantly increased phosphorylation of Λ‐methyl‐d‐aspartate receptor subunit NR2B as 6well as the activated forms of extracel‐lular signal‐regulated kinase and the cAMP response ele‐ment binding protein in SC. The increased levels of phosphorylation of these molecules, however, are signif‐icantly inhibited by the EphB receptor blocker. These findings indicate that EphB receptor signaling, probably by interacting with NR2B in SC, contributes to the devel‐opment of opioid physical dependence and withdrawal effects. This novel role for EphB receptor signaling suggests that these molecules may be useful therapeutic targets for preventing, minimizing, or reversing the devel‐opment of opiate dependence.—Liu, W.‐T., Li, H.‐C., Song, X.‐S., Huang, Z.‐J., Song, X.‐J. EphB receptor sig‐naling in mouse spinal cord contributes to physical depen‐dence on morphine. FASEB J. 23, 90‐98 (2009)