A simple, disposable, biphasic cultivation chamber has been developed for respiratory tract epithelial cells. This chamber, the Whicutt chamber, contains a movable, transparent, permeable gelatin membrane that can be employed either submerged in the culture medium, thereby feeding the cells by the traditional immersion method, or raised to the surface of the culture medium, to bring the apical surfaces of the cells into contact with air and provide nutrients only from below (basal feeding). The effects of biphasic cultivation on the growth and differentiation of respiratory tract epithelial cells from different sources have been studied in Whitcutt chambers. Primary hamster tracheal epithelial (HTE) cells grown to confluence with basal feeding developed a ciliated columnar morphology, with differentiated features (cilia and mucous granules) located in the apical region of the epithelial layer. These cells secreted mucinlike molecules from the apical surface (i.e. the surface in contact with air). Although the apical localization of differentiation features was greater, mucous cell differentiation achieved by basal feeding was quantitatively not greater than that achieved by continuous immersion feeding. Similarly, basal feeding did not alter the degree of epithelial cell differentiation in cultures derived from rat, rabbit, and monkey tracheas or from human bronchial and nasal tissues. In contrast, the differentiation of guinea pig tracheal epithelial cells in culture was significantly influenced by the feeding method employed. When fed basally, guinea pig tracheal epithelial cell cultures expressed various mucociliary functions with resemblance to mucociliary layers in vivo, whereas constantly immersed cultures seemed stratified and squamous. These results suggest that, at least for guinea pigs, the combination of feeding methods provided by the Whitcutt chamber can be used to achieve differentiated cultures of tracheal epithelial cells with a polarity of differentiation that is similar to that observed in intact airways in vivo.