OBJECTIVE Recurrences are still a challenge despite appropriate techniques in cholesteatoma surgery. This study was designed to evaluate the level of Ki-67 expression in recurrent cases of cholesteatoma. METHODS The study included 32 patients (18 males, 14 females; mean age 34 years; range 12 to 63 years) who underwent surgery for otitis media. Of these, 19 patients had cholesteatoma, and eight patients had recurrent cholesteatoma. Five patients who underwent tympanoplasty for chronic otitis media comprised the control group. All the patients with cholesteatoma underwent radical mastoidectomy. At surgery, tissue samples of cholesteatoma were taken and prepared for immunohistochemical staining. In controls, retroauricular skin samples were used. The two patient groups with cholesteatoma were compared with respect to Ki-67 expression. RESULTS Increased cellular proliferation was detected in both groups of cholesteatoma. No significant difference was found between two cholesteatoma groups with respect to Ki-67 staining (p>0.05). Compared to the controls, patients with cholesteatoma and those with recurrent cholesteatoma had significantly higher levels of Ki-67 staining (p<0.05 and p<0.01, respectively). CONCLUSIONS Our results suggest that, despite a higher degree of proliferation in recurrent cholesteatoma cases, treatment failures may be mainly associated with the surgical technique, accompanying infections, and the type of cholesteatoma.