Plastic embedded tissue can provide better antigenic preservation, lesser non-specific staining as well as better morphology, especially in high magnification. Several investigations regarding the cytokeratin expression have been performed to evaluate the possible origin of cholesteatoma and the difference between the cholesteatoma and normal epithelial tissue. However, there still remain some dabates in the exact origin of cholesteatoma. This study aimed to investigate the distribution pattern of cytokeratins in cholesteatoma and other epithelial tissues in order to speculate the origin of the cholesteatoma. Surgical specimens of cholesteatoma matrix, external auditory canal skin, postauricular skin, tympanic membrane and middle ear mucosa were obtained, and embedded in plastic resin. Using monoclonal antibodies specific for different cytokeratins and immunohistochemical staining, the distribution of CK 4, CK 8, CK 10, CK 13, CK 16, CK 17 and CK 18 was analyzed. Middle ear epithelium was characterized by the presence of CK 8 and CK 18, CK 10 and CK 17 were consistently expressed in cholesteatoma, external auditory canal and postauricular skin. CK 16 was expressed in basal layers of external auditory canal and postauricular skin and whole layers of cholesteatoma. The cytokeratin distribution was similar between the cholesteatoma and external auditory canal skin this result may suggest the possible epidermal origin of cholesteatoma.