Promoter hypermethylation of various tumor-related genes is extremely frequent in Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC). To investigate the significance of the promoter methylation in EBVaGC, we focused on one of the important proteins in the carcinogenesis of the stomach, E-cadherin. Methylation-specific PCR analysis (MSP) was applied to surgically resected gastric carcinomas, together with immunohistochemistry, PCR-based analysis of mutations and allelic loss, and site-specific MSP of E-cadherin gene. By MSP, nearly all of the carcinomas showed aberrant methylation of E-cadherin promoter in EBVaGC (21/22), and the frequency of this aberration was significantly higher than that in EBV-negative gastric carcinoma (GC; 45/81; p = 0.0003). According to immunohistochemistry of E-cadherin, the frequency of abnormal staining pattern in EBVaGC (87%) was comparable to that in the diffuse type (80%), but higher than that in the intestinal type of EBV-negative GC (47%). Promoter methylation was well correlated with abnormal staining pattern in EBVaGC, but not in EBV-negative GC. Neither mutation nor allelic loss of E-cadherin was observed in EBVaGC. Methylation status of E-cadherin within each carcinoma was heterogeneous as far as examined. Thus, in addition to the known association involving p16, we determined that promoter methylation-mediated silencing of E-cadherin gene was also closely associated with the development of EBVaGC, although it becomes heterogeneous within a given tumor along its progression. © 2003 Wiley-Liss, Inc.