The development of routine techniques for the isolation and in vitro maintenance of conducting airway epithelial cells in a differentiated state provides an ideal model to study the factors involved in the regulation of the expression of mucociliary differentiation. Several key factors and conditions have been identified. These factors and conditions include the use of biphasic culture technique to achieve mucociliary differentiation, the use of such stimulators as the thickness of collagen gel substratum, the calcium level, and vitamin A, and such inhibitors as the growth factors, epidermal growth factor and insulin, and steroid hormones, for mucous cell differentiation. Using the defined culture medium, the life cycle of the mucous cell population in vitro has been investigated. It was demonstrated that the majority of the mucous cell population in primary cultures is not involved in the replication of deoxyribonucleic acid (DNA). However, the mucous cell type is capable of self-renewal in culture, and this reproduction is vitamin A dependent. Furthermore, differentiation from nonmucous to mucous cell type can be demonstrated by adding back a positive regulator such as vitamin A to the "starved" culture. Cell kinetics data suggest that vitamin A-dependent mucous cell differentiation in culture is a DNA replication-independent process, and the process is inhibited by transforming growth factor-beta1.