Background: The pathogenesis of cholesteatoma remains unclear, despite several theories. Alterations in the density of mast cells positive for cluster of differentiation 117 protein (also known as CD117) can be critical to cholesteatoma formation, due to the effect on keratinocyte growth factor production. This study aimed to investigate the potential role of these mast cells in cholesteatoma pathogenesis. Methods: The number and density of mast cells positive for cluster of differentiation 117 protein were immunohistochemically analysed in 52 patients: 22 with chronic otitis media alone (group one), 25 with chronic otitis media with cholesteatoma (group two) and five controls. Results: The number of these mast cells was much higher in group two (in cholesteatoma matrix tissue) than in group one (in chronic otitis media granulation tissue) or the controls (in normal post-auricular skin). The density of these mast cells was significantly greater in group two than in group one or the controls (p < 0.05). The number and density of these mast cells was much greater in group one than in controls (p < 0.01). Conclusion: Mast cells positive for cluster of differentiation 117 protein could play a role in cholesteatoma formation. Further investigation of the role of these mast cells in cholesteatoma may suggest new ways of addressing this disorder, and may enable the development of targeted treatments. © 2012 JLO (1984) Limited.