Mucosubstance histochemistry of human middle ear cholesteatoma revealed that sialomucins are abundant and sulfomucins present in small amounts in the glandlike structures of the cholesteatoma perimatrix. Based on the study, various glycoproteins were injected into the dermis of the external ear canal and infiltrated into the tympanic membranes of guinea pigs. Tympanic membranes were obtained 7 days later and light- and electron-microscopically studied. Injection of a sialomucin from bovine submaxillary gland resulted in marked proliferation of epidermis and degeneration of the lamina propria. Asialomucin prepared from sialomucin by hydrolysis produced mild thickening of the epidermis but the lamina propria was not degenerated. Fetuin and bovine serum albumin did not cause proliferation of the epidermis. Cholesterol granuloma formed in some of the specimens injected with sialomucin. The presence of sialomucin in cholesteatoma perimatrix and these experimental studies using tympanic membranes of guinea pigs suggests that sialomucins participate in the proliferation of epidermis and degeneration of subepidermal connective tissue in human middle ear cholesteatoma.