Background: Acquired cholesteatoma is a complication of chronic otitis media that is usually associated with an intense local inflammatory reaction. Cholesteatoma probably arises from epithelial migration close to an ongoing host inflammatory response attributable to a chronic bacterial infection. Chlamydia pneumoniae is an intracellular microorganism associated with several pathologic conditions originally considered noninflammatory, including asthma, atherosclerosis, and Alzheimer disease. To investigate a possible relationship between C. pneumoniae and the development of cholesteatoma, tissue was studied in three different layers by polymerase chain reaction analysis. The results were compared with those relative to other two common middle-ear pathogens, Mycoplasma pneumoniae and Haemophilus influenzae. Methods: Cholesteatoma specimens were collected from 32 patients undergoing middle ear surgery. A series of 5-mum-thick specimens were obtained at three different tissue levels, internal (matrix), intermediate (perimatrix), and external (granulation tissue), and processed by polymerase chain reaction for detection of C. pneumoniae, H. influenzae, and M. pneumoniae. Fragmentation and polymerase chain reaction amplification were carried out using two substantially different techniques. Results: C. pneumoniae was detected with either polymerase chain reaction techniques in the internal layers in 16 of the 32 cholesteatomas (50%), associated with a positive finding in the intermediate layer in two cases and in the external layer in one case. Four specimens contained H. influenzae, always in the external layer, whereas none contained M. pneumoniae. Conclusions: The close relationship between cholesteatoma and C. pneumoniae demonstrated by the findings of this study could suggest a direct cause and effect link between the pathogen action and the clinical manifestations. Otherwise, a facilitated colonization by C. pneumoniae and chronic pathology of the ear could both take origin from a peculiar immunologic background of the host.