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Cholesteatoma is characterized by an excessive proliferation and differentiation of keratinocytes with a progressive accumulation of keratin debris. Caspase-14 is a novel regulator of keratinocyte terminal differentiation. The purpose of this study was to investigate the expression patterns and localizations of caspase- 14 in cholesteatoma and in normal external auditory canal (EAC) epithelium.The expression levels of caspase-14 mRNA were evaluated through real-time polymerase chain reaction (PCR) and Western blotting. Cholesteatoma and normal EAC epithelium were immunostained with a monoclonal antibody to caspase-14. The localizations of immunoreactivity to the caspase-14 antibody were compared between cholesteatoma and normal EAC epithelium through immunohistochemical staining.As shown by real-time reverse-transcription PCR, the expression level of caspase-14 mRNA in cholesteatoma epithelium was significantly higher than in normal EAC epithelium. Caspase-14 protein was detected in both normal EAC and cholesteatoma, but its expression was shown to be greater in cholesteatoma on Western blot analysis. As shown with immunohistochemical staining, caspase-14 protein was primarily expressed in the granular layer and the upper parts of the spinous layer in cholesteatoma epithelium and in the superficial layer of normal EAC epithelium. The expression of caspase-14 was more intense in cholesteatoma tissues than in normal EAC epithelium.The increased level of caspase-14 expression in cholesteatoma tissues may play a role in terminal differentiation of epithelium and accumulation of keratin debris from external matrix.