The air-filled cavity and ossicles of the mammalian middle ear conduct sound to the cochlea. Using transgenic mice, we show that the mammalian middle ear develops through cavitation of a neural crest mass. These cells, which previously underwent an epithelial-to-mesenchymal transformation upon leaving the neural tube, undergo a mesenchymal-to-epithelial transformation to form a lining continuous with the endodermally derived auditory tube. The epithelium derived from endodermal cells, which surrounds the auditory tube and eardrum, develops cilia, whereas the neural crest-derived epithelium does not. Thus, the cilia critical to clearing pathogenic infections from the middle ear are distributed according to developmental derivations. A different process of cavitation appears evident in birds and reptiles, indicating that this dual epithelium may be unique to mammals.