The aim of this study was to determine the distribution and apoptotic capacity in the various layers of cholesteatoma epithelium compared to the normal skin. Cholesteatomas were collected during surgical procedures of the ear. Normal skin specimens taken from the retroauricular area served as controls. Apoptosis was detected by using highly specific antibodies against APO2.7 antigen. The Membstain Apoptosis kit Direct based on in situ labeling of nuclear DNA fragmentation (Tolt-mediated dUTP nick and labeling TUNEL staining) was used. The distribution of apoptotic cells in the layer of the cholesteatoma epithelium and the epidermis was determined. The percentage of apoptotic cells was then counted per 100 cells of cholesteatoma epithelium and the normal epidermis in three different areas of each section and expressed in terms of mean +/- SD. An automatic analyzing system was used for counting. In normal epidermis, the apoptotic cells were observed in the granular layer of the epidermis. The percentage of these cells was 28.5 +/- 8.1%. A significantly greater number of apoptotic cells was observed in the suprabasal layers of the cholesteatoma epithelium (47.39 +/- 6.2%). A statistically significant difference was obtained for APO2.7-positive cells in the cholesteatoma epithelium as compared to the skin (P < 0.05) as determined by Student's t-test and the Mann-Whitney U test. Apoptotic activity is mainly observed in the suprabasal layer of cholesteatoma epithelium. It is not seen in the basal layer. Apoptosis plays an important role in the pathogenesis of cholesteatoma. It seems to be part of the differentiation and accumulation of keratin debris within the middle ear and expansion of cholesteatoma.