Substance P (SP)-, calcitonin gene-related polypeptide (CGRP)-, and vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) nerve fibers were observed in the epithelium of the canine subglottic region. To investigate the morphological differences among SP-, CGRP-, and VIP-IR nerve fibers in the epithelium, this study was performed by using immunohistochemical staining and electron microscopy, after which the origins of the nerve fibers were examined by denervation of the bilateral superior and inferior laryngeal nerves. SP- and CGRP-IR nerve fibers entered the epithelium through the basement membrane, ascended among the basal and ciliated cells, and reached under the epithelial junctional complex to terminate with varicosities. Furthermore, subsurface cistern-like structures or bouton en passant type synapse-like structures were observed among some varicosities of these nerve fibers and ciliated cells in the epithelial apical portion. On the other hand, VIP-IR nerve fibers entered through the basement membrane, and terminated with varicosities at the height of the middle portion of the subglottic epithelium. The varicosities of the VIP-IR nerve fibers were larger, and these varicosities had a greater number of synaptic vesicles than the SP- or CGRP-IR nerve fibers. After section of the bilateral superior and inferior laryngeal nerves, the SP- and CGRP-IR nerve fibers disappeared, while the VIP-IR nerve fibers were not affected. The density and distribution pattern of VIP-IR nerve fibers clearly differed from the SP- and CGRP-IR nerve fibers of sensory origin.(ABSTRACT TRUNCATED AT 250 WORDS)