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Background and Objectives:Retinoic acid (RA) can prevent keratin formation and induce mucous differentiation in epithelia. In the present study, we attempted to induce keratinizing squamous epithelium from human cholesteatoma epithelial (HCE) cells using an air-liquid interface (ALI) technique. We also examined the effect of RA on the phenotype of keratinizing HCE cells. Materials and Method:HCE cells were cultured in RA-free defined media at an ALI or in a submerged state. We phenotype after RA treatment. We also determined the effect of RA on the mRNA expressions of the cornifin-α and mucin genes as indicators of squamous and mucous diferentiation, respectively. Results:Using an ALI technique, we were able to diffe-rentiate HCE cells into a keratinizing squamous epithelium. When we treated the keratinizing HCE cells with RA, the morphological phenotype progresively changed to mucociliary epithelium. In addition, the expresion of cornifin-α mRNA was suppresed, and the expresions of mucin gene 5AC (MUC5AC) and mRNA were increased progressively with RA treatment. Conclusion:We sucesfully developed a culture system for keratinizing diferentiation of HCE cells using the ALI technique in a defined medium. Our study also clearly showed that RA treatment led to mucociliary differentiation of HCE cells. (Korean J Otolaryngol 2003 ;46 :727-32)