The aim of this study was to determine whether EBV associates with esophageal squamous cell carcinoma (ESCC), the most common malignancy in some parts of northern China, because these tumors frequently have an intense lymphocyte infiltrate. Fifty-one paraffin-embedded samples of ESCC from a high-risk area of North China were studied. The tumors included 9 well-differentiated, 31 moderately differentiated, and 11 poorly differentiated tumors. The cancer tissues and their nonmalignant adjacent mucosa (16 dysplastic and 42 normal) were evaluated by in situ hybridization using an antisense EBV-encoded RNA-1 probe and PCR amplification for EBV BamHI W fragment. In all cases, EBV was negative by both in situ hybridization and PCR. Our study suggests that EBV does not play a role in the carcinogenesis of ESCC in the geographic region.