AbstractHamster tracheal epithelial (HTE) cells maintained in primary culture show the induction of specific keratin species under vitamin A‐deficient conditions. A comparison was made between the morphology and the expression of keratins in HTE cells in vivo and in primary culture with and without vitamin A. HTE cells cultured in serum‐free, vitamin A‐supplemented medium formed a simple cuboidal, ciliated monolayer and produced four simple epithelial keratins (7, 8,18, and 19). In contrast, vitamin A‐deficient HTE cells, which were squamous‐like and stratified in culture, produced a more complex keratin pattern, with the induction of four additional keratin species (5,6, 14, and 17). A keratin pair whose expression serves as a marker of stratified epithelia was induced, as well as a single keratin species unique to lesions of squamous metaplasia in vitamin A‐deficient hamster tracheal organ cultures. Thus it appears that HTE cells retain the ability to respond to a deficiency in vitamin A through squamous differentiation and increased keratin production when removed from the intact organ and maintained in primary culture in a chemically defined medium. This system may be useful for the study of mechanisms underlying the squamous differentiation of respiratory epithelial cells in the development of bronchog