Squamous cell carcinoma of the middle ear (MESCC) is an uncommon tumor and is associated with a history of long-term chronic otitis media (COM) in most cases. Although the human papillomaviruses (HPVs) have been implicated in many human neoplasms, its role in the pathogenesis of MESCC has not been studied. Polymerase chain reaction (PCR) using consensus primers for the detection of HPV types 6, 11, 16, 18, 31, 33, 52b, and 58 was applied in screening fourteen cases of MESCC from archival material. Further subtyping was performed by restriction enzyme digestion of PCR products and by in situ hybridization method on tissue sections. Of the fourteen cases of MESCC, eleven were found to have HPV DNA. HPV-16 was detected in all positive cases. Five cases revealed both HPV-16 and -18. A history of long-term COM (> 3 years) was found in thirteen of the cases. This is the first report to localize HPV-16/18 in MESCC on both the tissue level and the molecular level. The high prevalence of HPV in COM-associated MESCC therefore provides a good model to explain the pathogenesis of chronic-inflammation-related human malignancies.