During the last decade middle ear epithelium has been cultured from various species. Until now, subcultivation has been achieved only with the use of a feeder-cell layer or conditioned medium. These factors are possible confounders in the in vitro model. On the other hand, subcultivation is necessary for exact quantitative studies. We present a reproducible culture method allowing subcultivation without feeder-cells or conditioned medium. The main features in our method are a low-serum, hormone-supplemented medium, an incubation temperature of 34 degrees C, fixation of explants, gentle trypsinization and replating with high cell density. Cells were identified by immunohistochemistry through a battery of monclonal antibodies. The percentage of epithelial cells in the subculture was 99.2%. To our knowledge, this is the first report describing subcultivation of middle ear epithelial cells exclusively in a completely controlled environment. These are optimal circumstances for future investigation and quantification of various factors influencing proliferation and differentiation of middle ear epithelium.