Abstract Conclusion: The detection of the HER4 receptor in 50% of cholesteatomas but never in the reference tissue, and the increased expression of its activating ligand EPI, suggest that EPI-mediated activation of HER4 might play a role in cholesteatoma growth. Objective: To investigate the expression of the epidermal growth factor (EGF) system in human middle ear cholesteatoma. Methods: Forty-seven patients referred for surgery due to cholesteatoma were included in the study. Clinical data were collected. Biopsies of cholesteatoma and skin from the external ear canal were obtained during surgery. mRNA expression was quantified with real-time PCR. The corresponding proteins were visualized using immunohistochemistry. Results: A systematic investigation of all four receptors, HER1, HER2, HER3, and HER4, and the ligands EGF, transforming growth factor (TGF)-α, amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin (EPI) of the EGF system is presented. At the mRNA level, the study demonstrates an up-regulation of mRNA encoding EPI and AR. In contrast HER1 and EGF were down-regulated. HER4 mRNA could be detected in 50% of cholesteatoma and 20% of reference tissues, and the HER4 protein was detectable only in cholesteatoma tissue. HER1 and HER2 were also visualized by immunohistochemistry, whereas the ligands EPI, AR, and EGF were undetectable with our methods.