The progressive growth of epidermis appears to be a crucial factor in the pathogenesis of cholesteatoma. How this growth is encouraged is still not well understood. In the present study, lymphotoxin was found in human middle ear cholesteatoma tissues by the immunoperoxidase method using rabbit anti‐human lymphotoxin immunoglobulin G (IgG). Lymphotoxin was localized in the epithelium and connective tissues of the cholesteatomas studied. The epithelium of normal external ear canal skin was lightly stained, but the stain was much weaker than that of the cholesteatomas. In vitro, recombinant human lymphotoxin stimulated proliferation, protein synthesis, and terminal differentiation of basal keratinocytes. These findings suggest that lymphotoxin may be one of the mediators involved in the development of cholesteatoma.