It is strongly suspected that the Epstein-Barr virus (EBV) plays a role in the genesis of nasopharyngeal and gastric carcinoma. The aim of this study was to search for such a connection between esophageal squamous cell carcinoma (ESCC) and EBV. We investigated 104 surgically resected esophageal cancers using in situ hybridization (ISH) for EBV-encoded RNA (EBER). We found no EBER-positive cancer cells in any tests, although there were five samples in which EBER-positive tumor-infiltrating lymphocytes (TILs) were found. We conclude from this study that EBV is not associated with ESCC.