To examine whether human papillomavirus (HPV) DNA is associated with esophageal cancer, frozen and paraffin-embedded neoplasms of the upper aerodigestive tract, including esophageal cancer, were investigated. DNA obtained from frozen specimens and cell lines were analyzed by both polymerase chain reaction (PCR) and Southern blot hybridization. DNA from paraffn-embedded samples were analyzed strictly by PCR. DNA of HPV types 6 and 11 was detected in papillomas of the upper respiratory tract at >50%. However, HPV DNA was infrequently detected in specimens from the upper digestive tract (31 esophageal cancers and 2 esophageal carcinoma-derived cell lines), even by PCR at a sensitivity of 0.1 copy number per cell. These results suggest that the etiologic significance of HPV infection in esophageal cancer is negligible.