The CDKN2A gene encodes a cyclin-dependent kinase inhibitor, p16, which promotes cell cycle arrest. Methylation of the promoter region of the gene transcriptionally inactivates the gene. We have analyzed the methylation status of the promoter region of the CDKN2A gene in gastric adenocarcinomas using methylation-specific polymerase chain reaction. We also examined the tumors by immunohistochemistry for p16 protein. Of 114 gastric adenocarcinomas analyzed by immunohistochemistry, 34 cases (30%) were negative for p16 protein. Twenty-four of these 34 cases (71%) had methylation of the promoter region of the CDKN2A gene. Methylation of the promoter was strongly associated with loss of p16 protein by immunohistochemistry (P <0.0001). Neither stage, grade, anatomic site, or histologic subtype of the tumor nor age, gender, ethnic origin, or survival time of the patient were significantly different between the groups characterized by tumors with and without methylation. CDKN2A promoter methylation was not significantly associated with microsatellite instability.