The role of high-risk human papillomavirus (HPV) in the pathogenesis of esophageal squamous cell carcinoma (ESCC) remains unclear. p16(INK4) is used as a surrogate marker to detect HPV-related tumors but has had discrepant results in ESCC. In this study, 32 cases of ESCC were examined to determine the relationship between p16(INK4) expression and high-risk HPV. All the tumors were stained by immunohistochemistry for p16(INK4). Tumors having p16(INK4) nuclear and/or nuclear and cytoplasmic expression were considered positive. Tumors positive for p16(INK4) expression were tested for high-risk HPV by in situ hybridization (ISH). In all, 20 cases of ESCC (63%) showed only cytoplasmic staining for p16(INK4), and 11 cases (34%) showed both cytoplasmic and nuclear staining for p16(INK4); 4 cases (13%) showed no staining for p16(INK4). None of the p16(INK4) -positive cases were positive for high-risk HPV by ISH. These results indicate that p16(INK4) expression in ESCC does not correlate with the presence of high-risk HPV DNA by ISH. High-risk HPV does not seem to play a major role in the carcinogenesis of ESCC in low-risk areas.