Transglutaminase (TGase) isoenzymes are involved in the process of the differentiation and cornification of keratinocytes in the epidermis. This study investigates the presence and localization of three TGase isoenzymes to elucidate the nature and differentiation status of the squamous epithelium in human aural cholesteatoma. Twenty cholesteatoma specimens were used. The presence and localization of three TGase isoenzymes were studied by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. mRNA expression of three TGase isoenzymes were detected in the tested cholesteatomas with variable levels. The immunohistochemical staining patterns of three TGase isoenzymes showed variations within specimens, relating to keratinizing activity. TGase K is the most abundant among three isoenzymes. Keratinizing epithelium of cholesteatoma have similar expression profiles of TGase isoenzymes with those of epidermis of the skin. Other areas, particularly those showing non-keratinizing epithelium, showed weak immunostaining of TGase E and C, suggesting its different maturation status from keratinizing epithelium. The results of this study indicate that epithelium of cholesteatoma undergoes same direction of maturation and differentiation characteristics as the epidermis of skin, evidenced by similar expressions of TGases both in mRNA level and immunohistochemistry.