Cholesteatoma is a disease of the temporal bone with invasion of squamous epithelium into the middle ear cavity. There has been considerable research for cholesteatoma in recent years but an understanding of the pathophysiology has been handicapped by the lack of a suitable research model. Patients undergoing tympanomastoid surgery for cholesteatoma were sought and cholesteatoma matrix from the middle ear cavity was obtained, as well as skin from both parts of external auditory canal and postaural skin. Culture medium of Medium 154(Cascade Biologics Inc., USA) and HKGS(Cascade Biologics Inc., USA) was used for keratinocyte culture in serum-free media, in the absence of feeder layer, and in the absence of contaminating fibroblasts. Morphological differences of cholestestoma was compared with postauricular skin. The keratinocytes of normal skin appeared darker and more granular than that of cholesteatoma matrix. Also there was a delay in formation and growth of colonies in keratinocytes of cholesteatoma. In present study, the authors established primary culture of keratinocytes of cholesteatoma in serum-free medium for the first time in the literature review. Thus this could be successfully employed for in vitro studies of human cholesteatoma pathophysiology.