Human papillomavirus (HPV) infection, especially high risk types HPV 16 and 18, have been studied widely in cervical cancer. However, HPV infection in esophageal cancer has not been well defined. In the present study, immunohistochemistry, PCR and Southern blot hybridization methods were used to detect HPV infection in 127 cases of esophageal squamous cell carcinoma. Immunohistochemistry results indicated that the virus was detected frequently in well differentiated carcinoma. The positive rates for BPV and HPV E6 protein were 60.6% (77/127) and 43% (54/127) respectively. Meanwhile, PCR and Southern hybridization showed that 35.9% (37/103) of esophageal squamous cell carcinomas have HPV DNA, which included 20.4% (21/103) HPV 16 and 7.8% (8/103) HPV 18. Of the 103 cases, only 1 had both HPV 16 and HPV 18 DNA. Our results suggest that HPV infection is present in esophageal squamous cell carcinoma and may play a role in its pathogenesis.