When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of airway epithelial biology and differentiation. We have performed microarray analysis on ALI cultures of human bronchial epithelial cells (HBECs) grown over a 28-d period to identify genes involved in mucociliary differentiation. We identified over 2,000 genes that displayed statistically significant 2-fold or greater changes in expression during the time course. Of the genes showing the largest increases, many are involved in processes associated with airway epithelial biology, such as cell adhesion, immunity, transport, and cilia formation; however, many novel genes were also identified. We compared our results with data from proteomic analyses of the ciliary axoneme and identified candidate genes that may have roles in cilia formation or function. Gene networks were generated using Ingenuity Pathways Analysis (Ingenuity Systems, Redwood City, CA) to identify signaling pathways involved in mucociliary cell differentiation or function. Networks containing genes involved in TGF-beta, WNT/beta-catenin, and epidermal growth factor receptor (EGFR) pathways were identified, suggesting potential roles for these families in airway epithelia. Microarray results were validated by real-time RT-PCR for a number of representative genes. This work has provided extensive information about gene expression changes during differentiation of airway epithelial cells, and will be a useful resource for researchers interested in respiratory function, pathology, and toxicology.