To elucidate the association of human papillomavirus (HPV) and Epstein-Barr virus (EBV) with carcinogenesis of the esophagus, 41 surgically resected specimens and 12 cell lines of esophageal squamous cell carcinoma were examined for the presence of HPV DNA and EBV DNA by polymerase chain reaction using primers for the E6 regions of HPV-16 and -18 and for the EBNA 1 region of EBV. We designed the reaction condition to amplify HPV and EBV DNA specifically and detected by gel electrophoresis. In ethidium bromide staining, no band was detected either for the HPV E6 region of for the EBV EBNA 1 gene in any of the surgically resected specimens and the cell lines, although the HPV sequence was detectable by Southern blot hybridization, which is a more sensitive detection method than staining; three out of 41 surgically resected specimens were positive for HPV-18 by Southern blot hybridization of polymerase chain reaction products. However, the number of viral genomes has been estimated as lower than 1 x 10(-3) copies per cell based on the intensity of the hybridization signals. Moreover, the DNA samples extracted from the corresponding non-cancerous esophageal mucosa were also positive for HPV-18, and the intensities of the hybridization signals were almost the same as those of the tumors. The results of our study indicate that HPV-16, HPV-18 and EBV are not generally associated with esophageal carcinogenesis.