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Primary cultures of human airway bronchial airways represent a valuable tool in understanding the roles of the epithelium, cilia, and the mucus layer in coordinating the clearance of mucus from the airways. The ability to obtain cells from both normal and diseased populations (such as cystic fibrosis and Chronic obstructive pulmonary disease (COPD)) allows researchers to investigate the disease phenotype on these processes. Furthermore, such cultures have provided investigators with a vast source of native airway mucus, devoid of external biological processes that occur in vivo, for biochemical and rheological studies. The primary goal of this chapter is to describe the culturing and use of human airway cultures grown under an in vivo-like air–liquid interface for use in a variety of mucus and mucociliary studies.