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Cholesteatomas are frequent middle ear benign tumors of unknown etiology. Infectious agents have been considered as possible contributing factors in the pathogenesis of cholesteatomas. Aiming to investigate the presence of respiratory viruses in primary cholesteatoma tissues, 26 formalin‐fixed paraffin‐embedded primary cholesteatoma tissues obtained from patients seen at the of the Clinical Hospital of the University of São Paulo School of Medicine, in Ribeirão Preto, Brazil were tested by real‐time polymerase chain reaction (PCR). Considering the PCR results, 35% of the tissues were positive for human rhinovirus (HRV), 15.3% for human enterovirus (EV), 3.8% for human metapneumovirus (HMPV), and 3.8% for human bocavirus (HBoV). Serial immunohistochemistry for virus antigens and cell surface markers evidenced that the viruses were associated with fibroblasts, dendritic cells, macrophages, B lymphocytes, CD4+, and CD8+ T lymphocytes. These findings indicate for the first time the presence of active respiratory virus infection in primary cholesteatoma tissues, suggesting that persisting virus infection in the middle could play a role in the pathogenesis and evolution of cholesteatomas. Highlights: Primary cholesteatoma tissues are sites of respiratory virus infections.Genomes and proteins of HBoV, HMPV, HEV and HRV were detected in primary cholesteatomas.Viral antigens and cell surface markers evidenced that infected cells were fibroblasts, dendritic cells, macrophages, B lymphocytes, CD4+ and CD8+ T lymphocytes. [ABSTRACT FROM AUTHOR]