Proliferative sites within the overgrowing epithelium of cholesteatoma are still ill defined. In this study, we used the monoclonal antibody Ki-67 on frozen cut sections from 23 cholesteatoma samples obtained at surgery from 20 patients. This reagent has been reported to stain the nucleus of proliferating tumor cells and the cytoplasm of growing keratinocytes. In this series of samples, flat superficial layers of epithelium were consistently negative for Ki-67 or displayed a faint staining of the basal layer. By contrast, the deepest areas of epithelial recesses appeared brightly stained with Ki-67. The latter were often in the close vicinity of inflammatory cells present in the underlying mucosa. This observation suggests that cholesteatoma growth is initiated within the deep epithelial folds of overgrown tympanic skin, and that it might be triggered or sustained by inflammatory cytokines.