Frozen sections of cholesteatomas were compared with postauricular and auditory canal skin and studied using histochemical methods. Several dehydrogenases, lysosomal enzymes and proteolytic activity were studied. Lactate and malate dehydrogenase activity was very strong in the epithelium and subepithelial tissue of cholesteatomas; no succinate dehydrogenase activity was observed. Esterase and acid phosphatase activity was prominent in cells of granulation tissue. Considerable proteolytic activity was observed in these cells and one specimen showed possible extracellular activity. Strong evidence of fat was found in the granulation tissue, particularly at the interface of granulation tissue and bone. In postauricular and auditory canal skin, enzyme activity was generally weaker; no evidence of fat was found. The findings are discussed in the light of other investigations on the importance of enzyme activity in bone destruction associated with cholesteatoma.