Gastrin-releasing peptide (GRP) is widely distributed in the central nervous system, and is involved in a variety of physiological processes and behaviors, including the circadian rhythm, food intake, anxiety/fear responses, and male sexual function. Recently, the spinal GRP/GRP-receptor system has been identified as an itch-specific mediator in the somatosensory system. We demonstrated that the expression of GRP was predominant in the small-sized neurons of the primary afferents, and GRP-immunoreactive axons terminated the superficial layers of the spinal dorsal horn in rats. Furthermore, ultrastructure of GRP-immunoreactive axon terminals was analyzed by various kinds of electron microscopy. The analysis of high-voltage electron microscopy showed that GRP-immunoreactive axon terminals formed a series of the varicosities. The analysis of transmission electron microscopy showed the GRP-immunoreactive presynaptic terminals contained many clear microvesicles and large dense-cored vesicles. Serial block-face scanning electron microscopy (SBF-SEM) that combines SEM and ultramicrotome makes serial ultrathin sections automatically. Until now was technically impossible, but the reconstruction of a three-dimensional ultrastructure has become possible owing to this electron microscopy.We have developed a technology that applies immunohistochemistry to label the target molecule in this microscope. SBF-SEM analysis showed that not one but some dendritic spines contacted a single varicosity containing GRP-immunoreactive dens-cored vesicles. Thus, by combination of several kind of electron microscopy, visualization of a three-dimensional ultrastructure of neuropeptide in the axon terminals has become possible.