μopioid receptors (MOR) are internalized into endosomes after binding to agonists. Previous studies have suggested that MOR internalization followed by receptor recycling can contribute to the prevention of opioid tolerance; however, the relevance of MOR internalization to opioid tolerance remains controversial. Our recent study used immunoelectron microscopy to clearly show the intracellular localization of MOR in spinal cord dorsal horn neurons after in vivo exposure to two different opioids: [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and morphine. MOR immunoreactivity was scattered within the perykaryon after exposure to DAMGO, often in close apposition to lysosomes and Golgi cisterns. In contrast, MOR immunoreactivity was localized on the cell membrane after exposure to morphine. Fentanyl, a clinically important MOR agonist, is commonly used for various types of pain relief. Although the development of tolerance to fentanyl is a major clinical problem, little information is available regarding the morphological aspects of fentanyl-induced MOR internalization, especially at the electron microscopic level. Therefore, in the present study, we investigated MOR internalization in dorsal horn neurons after fentanyl treatment using immunoelectron microscopy. Under anesthesia, each rat was prepared with intrathecal catheter insertion at the L4-5 level, followed by intrathecal injection with fentanyl (10 μg/10 μl). After exposures to the agent, animals were subsequently perfused with a fixative solution, after which their spinal cords were extracted. A vibratome was used to obtain transverse sections (40 μm) of the lumbar spinal cord; the sections were processed using both pre-embedding immunoperoxidase and immunogold-silver enhancement methods. MOR immunoreactivity in the laminae II neurons of the dorsal horn was observed using an electron microscope. MOR immunoreactivity was detected within the perykaryon but not on the cell membrane five min after exposure to fentanyl, suggesting that fentanyl induces more rapid internalization of MOR compared to other opioids.