AIM OF THE STUDY: Tympanosclerosis is a middle ear disease located in submucous membrane. It leads to the tympanic membrane and ear ossicles fixation which results in severe conductive hearing impairment. The etiology of tympanosclerosis remains unclear. Cytokines play very important role in development of tympanosclerosis. The research aimed to assess gene expression level of pro inflammatory cytokine TNF-α in selected structures of middle ear in patients with chronic otitis media with tympanosclerosis. METHODOLOGY: The research was performed on middle ear tissues with tympanosclerosis achieved from 30 patients operated on in the Otolaryngology Department of the Medical University of Gdańsk. The control group was formed by tympanic membranes sampled from 17 corpses of patients who died suddenly. Advancement of tympanosclerotic changes were grouped based on Tos classification. The TNF-α gene expression level was measured by real time PCR with specific hydrolysed probes (TaqMan). The gene expression level for TNF-α was correlated with clinical classification of tympanosclerosis. RESULTS: The level of gene profile expression for TNF-α in selected structures of the middle ear with tympanosclerosis was statistically significant, higher in comparison with the control group. The highest level of expression was observed in group two which is relevant to more advanced tympanosclerosis. CONCLUSION: Tympanosclerosis is a result of constant inflammatory process and it is modulated by cytokines including TNF-α. The positive correlation between gene expression level of TNF-α and tympanosclerosis could in the future contribute to antiinflammatory, medical treatment of tympanosclerosis.