Several studies have documented the frequent occurrence of human papillomavirus (HPV) DNA in esophageal squamous cell carcinomas (ESCC) in patients from geographic regions where the incidence of this type of cancer is high, such as parts of China. However, the prevalence of HPV infection in ESCC in patients from low incidence geographic regions, such as North America, remains controversial. Therefore, this study evaluates the prevalence of HPV in ESCC in patients from North America, a region where the population is considered at low risk for the development of this neoplasm. ESCCs in 51 patients from three North American cities were analyzed for the presence of HPV DNA by a highly sensitive and specific polymerase chain reaction (PCR) method. Tumor DNA was extracted from formalin-fixed, paraffin-embedded tissue specimens and assayed by PCR using an L1 HPV consensus sequence primer, as well as HPV 16 and HPV 18 E7 region primers. The use of consensus primers to the L1 region allows for detection of most known HPV types and many novel HPV types. Appropriately sized reaction products were analyzed by restriction fragment length polymorphism (RFLP) to confirm the presence and type of HPV, and to exclude products produced by amplification of human DNA sequences. After complete analysis, only one case (2%) of ESCC was HPV DNA positive. This case was independently confirmed using L1 and E7 consensus primers as HPV type 16 and was the only case that tested positive with either assay. These results show that, in contrast to geographic regions where ESCC is prevalent, HPV infection occurs infrequently in association with ESCC in patients from North America.