Recent studies, mainly from high risk areas of oesophageal cancer (OC), have suggested a possible role of oesophageal infection with human papillomavirus (HPV) in the carcinogenesis of OC. Twenty-two cases of OC (among them 20 cases of squamous cell, 1 case of verrucous and 1 case of adenoid cystic carcinoma) originating from a middle risk area of OC, were analysed by in situ hybridization (ISH) and polymerase chain reaction (PCR) to demonstrate HPV infection. No evidence of HPV DNA was found using ISH. By PCR, the presence of HPV DNA was detected in 1/3 (33%) of early and 1/17 (5.7%) of advanced squamous cell carcinoma cases. After digestion of PCR products using seven restriction endonucleases, the restriction enzyme digestion patterns were found to be unique for HPV type 16 in both positive cases of OC. All other squamous cell carcinomas, the case of verrucous carcinoma and that of adenoid cystic carcinoma were successfully amplified only with internal control human beta-globin primers. Our results are comparable with those from high risk areas of OC and support the HPV may be involved in the carcinogenesis of this malignancy.