Mast cells (MCs) in rat airways have been classified into two subtypes: epithelial MCs and connective tissue MCs (CTMCs). However, the immunohistochemical characteristics, cellular morphology, and distribution of epithelial MCs in the upper airways remain unclear. The present study investigated the morphological characteristics and distribution of epithelial MCs using 5-hydroxytryptamine (5-HT) and other immunohistochemical markers in sectioned or whole-mount preparations of the rat larynx and trachea. A double immunofluorescence analysis revealed the co-localization of 5-HT immunoreactivity with c-kit, a stem cell factor receptor commonly used as a MC marker, in both epithelial MCs and CTMCs. Dopa decarboxylase, an enzyme involved in 5-HT synthesis, was detected in both subtypes, suggesting their ability to synthesize and release 5-HT. Tryptase and histidine decarboxylase (a biosynthetic enzyme of histamine), which are well-known mediators of MCs, were exclusive to CTMCs. Epithelial MCs were pleomorphic with long cytoplasmic processes, whereas CTMCs were round and lacked cytoplasmic processes. The density of epithelial MCs was significantly higher in the glottis and cranial part of the trachea than in the epiglottis and other parts of the trachea. The present results showed that the morphology and immunohistochemical characteristics of epithelial MCs were different from those of CTMCs in the rat larynx and trachea, and variform epithelial MCs were predominantly located at the entrance of the upper airways. Epithelial MCs may release 5-HT in the vicinity of epithelial cells that regulate innate immune responses by modulating epithelial cell functions at the entrance gate of the upper airways.