To date, more than 120 total types of HPV have been identified, and in a recent meta-analysis, HPV was confirmed as an independent risk factor for oral carcinoma. Some investigators have reported that HPV infection is related to certain factors, including the gender, age, alcohol consumption, smoking habit, sexual behavior and denture wearing. Denture wearing can lead to denture epulis, which is a hyperplasia of fibrous connective tissue caused by denture irritation. Recently, HPV infection was detected in the hyperplastic epithelium of denture fibroma, but it still has received little study. The objective of the present investigation, therefore, was to clarify the relationship between the hyperplastic epithelium of the denture epulis and HPV infection. DNA of 118 formalin-fixed, paraffin-embedded hyperplastic epithelium biopsies of epulis specimens was extracted. Firstly, HPV infection was detected by PCR using consensus primers. Secondary, PCR using HPV type-specific primers (low risk types 6 and 11; high risk types 16, 18 and 33) was done in positive PCR samples. HPV infection was also detected by in situ hybridization and immunohistochemical techniques. Eighteen (23.1 %) of the 78 cases of denture epulis were positive, and 2 (5.0 %) of the 40 cases of non-denture epulis were positive. The difference was statistically significant (p<0.05, using Fisher's exact test). The most frequent type of HPV found in the 14 positive samples was HPV 16. It seems that the hyperplastic epithelium of denture epulis is easily infected with viruses because the epithelium is exposed daily to traumatic irritation from dentures. These results suggest that the hyperplastic epithelium of denture epulis might be an important reservoir for HPV infection of the oral region where later HPV-associated diseases, such as oral cancer and other oral lesions, may develop.