Immunolocalization of involucrin in human middle ear cholesteatoma was performed with specific rabbit anti-human involucrin antibody. Involucrin was noted prominently in the cytoplasm of the suprabasal cells, including spinous cells and granular cells. The pattern of distribution of involucrin in cholesteatoma shows its epidermal characteristics. Immunostaining of involucrin was also made in tympanic membrane and external ear canal skin. As for comparing its distribution, a larger amount of involucrin was present in cholesteatoma (60%) than in external ear canal skin (29%) in our study and in normal skin (25%) in another study. This suggests active differentiation of the epithelial cells in cholesteatoma. Further investigation on the inhibition of involucrin formation may have some effect in the treatment of cholesteatoma.