Confluent monolayers of epithelial cells from a rabbit's tracheal mucosa is obtained via several methods of primary cultures, and characterization of the monolayers is discussed in terms of morphology and electrical resistance as follows: The tracheal epithelial (TE) cells are isolated with little damage under the condition that the trachea was immersed in the type XIV protease of 0.1%. In the cell culture, a serum-free hormone-supplemented medium is used for the growth of TE cells with cell culture inserts. The cilia and desmosome are observed in the monolayers cultured for 10 days; the TE cells were seeded on a collagen-coating membrane, and the mucosal side of the monolayers was out of the medium. The data of electrical resistance of monolayers indicates that a suitable duration is 9-10 days for the cell culture to construct a monolayer of the real tracheal epithelium.