We discuss several techniques of culturing middle ear epithelium from several species, which was formerly difficult but is now possible because of recent advances in culture methods. The three main methods of initiating a culture are organ culture, primary explant culture, and cell culture. Although each method has both advantages and disadvantages, investigators can choose the method most suitable to their purpose. A few researchers, including ourselves, have succeeded in obtaining serial culture of middle ear epithelium. Using 3T3 feeder layer technique or conditioned medium enabled us to develop a fibroblast-free cultured middle ear epithelium. The availability of cultured middle ear epithelium provides a potential tool in future middle ear research.