Aural cholesteatoma is characterized by invading squamous epithelia with altered growth properties. Cytokeratin (CK) expression is affected in epidermal proliferative diseases and represents the alterations of keratinocyte proliferation, differentiation, and migration. In the present study, the intensity of CK immuno-expression was determined, using densitometry at various sites in experimental cholesteatoma in order to characterize changes of keratinocytes. With cholesteatoma formation, CK4, a marker for non-keratinizing epithelia, increased in the suprabasal layers of the annular external auditory canal (EAC) and at the pars tensa indicating an altered differentiation and migration of keratinocytes. CK5/6, a marker of keratinizing squamous epithelium, increased only at the pars tensa of the tympanic membrane, indicating basal keratinocyte hyperplasia. CK1/10 increased in the suprabasal layer at the annular EAC, and at the peripheral pars tensa, indicating increased terminal differentiation of keratinocytes. CK13/16, markers of differentiation and hyperproliferation, increased in suprabasal layer of the EAC, and at the peripheral pars tensa. However, it decreased in the basal layer of the EAC, indicating hyperproliferation and migration of keratinocytes. The findings of this study support the basal cell hyperplasia hypotheses for the pathogenesis of aural cholesteatoma, with regard to hyperproliferation, migration, and an altered differentiation of keratinocytes.