Recent technical advances now permit the serial culture of normal human middle ear epithelial (NHMEE) cells. However, the ciliary differentiation of these cells has not been achieved. The purpose of this study was to establish a culture system in order to differentiate serially cultured NHMEE cells into ciliated cells. If ciliated cells developed, the percentages of ciliated cells and secretory cells were measured throughout the duration of culture. We also examined the levels of mucin and lysozyme secretion and their mRNAs in a time-dependent manner. Human middle ear mucosa with a normal appearance was harvested and serially cultured after enzymatic disaggregation. These cells were cultured in an air-liquid interface (ALI) culture system for 2, 7, 14, 21 and 28 days after confluence. Ciliogenesis usually began 16-18 days after confluence. The percentage of ciliated cells detected by means of immunohistochemical staining increased over time up to a maximum of 10.6% but the percentage of secretory cells remained stable at approximately 40% throughout the duration of culture. By Day 14 after confluence, the amounts of mucin and lysozyme secretion, as measured by dot-blotting analysis, had increased significantly and then remained stable. The expression levels of mucin gene 5B (MUC5B), MUC8 and lysozyme increased with the duration of culture. MUC8 in particular showed a dramatic increase on Day 28 after confluence. In contrast, the level of MUC5AC mRNA peaked on Day 14 after confluence, and then decreased. In conclusion, ciliary differentiation of NHMEE cells can be induced using an ALI culture system. Our study also suggests that secretory function develops earlier than ciliogenesis, and that the expressions of MUC5B and MUC8 mRNAs increase as a function of differentiation.