Cholesteatomas are histologically benign, though biologically invasive lesions that arise from the migration of squamous epithelium of the ear. Acquired Cholesteatomas usually arise in an antigenically active environment, i.e., a chronically and/or recurrently inflamed middle ear. In contrast, congenital Cholesteatomas occur in an uninflamed environment. The potential role of dendritic cells (DCs) in the evolution of this lesion has not been thoroughly studied. By staining for S-100 protein, the authors evaluated the presence and distribution of DCs in Cholesteatomas. Sixteen cases of Cholesteatomas diagnosed from 1987 to 1989 were selected for this study. The formalin-fixed, paraffin-embedded sections were processed by a standard avidin-biotin peroxidase- antiperoxidase method for S-100 protein and for leukocyte common antigen (LCA). The presence and distribution of S-100 protein-positive DCs was evaluated and compared to canal wall skin. DCs were present in all cases. Nine acquired Cholesteatomas had 5 to 16 epithelial DCs per high-power field (HPF). Seven congenital Cholesteatomas were examined. Four with isolated congenital pearl-like Cholesteatomas had 1 to 3/HPF epithelial DCs. In contrast, the three inflamed congenital Cholesteatomas had 6 to 12/HPF DCs. The control uninflamed canal wall skin had only 1 to 3/HPF DCs. All DCs were LCA negative, as expected.