Recent progress in cell culture enables us to grow and to maintain differentiated epithelial cells in a serum-free defined culture environment. Such an epithelial cell culture system free from interference by other nonepithelial cell types should be used widely in studies related to toxicology, carcinogenesis, and disease-related problems. This approach will lead to a better understanding of pathological changes indicated in the injured epithelial layer. The gap of information existing between in vivo and in vitro can be bridged together by this simple epithelial cell culture system by carefully analyzing changes of cell properties from in vivo to in vitro and by the cell separation to enrich specific cell types in preparation. Furthermore, evidence has been accumulated suggesting that the properties of epithelial cells in culture are part of the integral cellular physiologies of epithelial cells in vivo. These properties are, in most cases, related to cell injury which is reparable even in vitro if the appropriate condition is provided. Using the airway epithelial cell culture system developed in our laboratory as an example, the above points are discussed. Finally, we have shown that a differentiated tracheal epithelium with a similar polarity as in vivo was established in the described serum-free culture condition.