Virtually all in vitro studies of the effects of rhinovirus on human airway epithelium have used cells grown under conditions known to produce low levels of differentiation. The relevance of the results to native epithelium is questionable. Here we grew primary cultures of human tracheal or nasal epithelium under three conditions. One condition produced pseudostratified, mucociliary cells virtually indistinguishable from native epithelium. The other two conditions produced undifferentiated squamous cells lacking cilia. Cells were infected for 6 h with rhinovirus-16. After a 24-h incubation period, we determined levels of viral RNA in the cells, numbers of infectious viral particles released in the mucosal medium, expression of a variety of epithelial cytokines and other proteins, release of IL-6 and IL-8, and transepithelial electrical resistance and voltage. After infection, levels of viral RNA in the poorly differentiated cells were 30 or 130 times those in the differentiated. Furthermore, expression of mRNA for inflammatory cytokines, release of infectious particles, and release of IL-6 and IL-8 were closely correlated with the degree of viral infection. Thus well-differentiated cells are much more resistant to viral infection and its functional consequences than are poorly differentiated cells from the same source.