In the present study, the development of the secretory function of the murine tubotympanum, aging from gestational day 13 to postnatal day 21, was investigated using alcian blue/periodic acid-Schiff staining and lysozyme and lactoferrin immunohistochemistry. The secretory activity of the tubal epithelial cells was first observed at gestational day 17. Although tubal glands began to develop at gestational day 18, their secretory activity was first shown at postnatal day 3. The secretory activity of tubal epithelial cells rapidly increased immediately after birth, while that of the tubal gland showed gradual increment from the third postnatal day. Lysozyme was first recognized in the epithelial secretory cells at postnatal day 1, while lactoferrin was first detected in the tubal glands at the third postnatal day. The results indicate that the epithelial secretory cells in the murine tubotympanum, are mature at birth, but the tubal glands mature gradually after birth. In addition, the secretion of lysozyme and lactoferrin seemed to be related to the maturation of the secretory cells in the murine tubotympanum.