Human airway epithelial (hAE) cell cultures are instrumental for studying basic and applied aspects of respiratory tract biology, disease, and therapy. When primary epithelial cells from the human nasal passages or tracheo-bronchial airways are grown on porous supports at an air-liquid interface (ALI) they undergo mucociliary differentiation, reproducing both the in vivo morphology and key physiologic processes. These cultures are useful for studying basic biology, disease pathogenesis, gene therapy and aerosol administration of drugs. This chapter gives detailed protocols for tissue procurement, cell isolation, production of complex media, and cell culture initiation and maintenance needed for hAE cell ALI cultures with non-proprietary reagents.