To investigate whether--in cholesteatoma epithelium--terminal differentiation, resulting in high involucrin expression, is associated with mitogen-activated protein kinase (MAPK) signaling. Alterations in specific signal transduction pathways may explain abnormal differentiation of the keratinocytes in cholesteatoma. Signaling pathways used by eukaryotic cells to transduce extracellular signals into cellular responses converge on activated mitogen-activated protein kinases, mainly extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38. Tissue samples were taken from 16 patients with acquired cholesteatoma. Histologic examination showed that 12 of the 16 cholesteatomas were inflamed. Immunohistochemical methods were used to determine expressions of involucrin and the activated form of p38, extracellular signal-regulated kinase, and c-Jun NH2-terminal kinase proteins. The results obtained from cholesteatoma tissue were compared with paired control samples from retroauricular skin. We demonstrated increased levels of involucrin and increased levels of the activated forms of p38 and ERK1/2 in cholesteatoma epithelium when compared with control samples. No abnormality was found in the activation and expression of JNK1/2. A positive correlation was found between p38, pERK1/2, and involucrin expression (p<0.05). Our results demonstrate that signaling via the mitogen-activated protein kinases ERK1/2 and p38 is increased in cholesteatoma epithelium when compared with control skin. The correlations between involucrin-and phosphorylated pERK1/2 expression and between involucrin-and phosphorylated p38 expression indicates that terminal differentiation in cholesteatoma epithelium proceeds via activation of these mitogen-activated protein kinase signaling pathways. We discussed whether this increased mitogen-activated protein kinase-driven terminal differentiation is probably part of a keratinocyte survival program or caused by an inflammation-induced cellular stress response.