Aberrant promoter methylation is an important mechanism for gene silencing. In the present study, 50 Barrett's esophagus-associated esophageal adenocarcinomas (ADC), 50 cardiac ADC and 50 gastric ADC were investigated by means of methylation-specific real-time PCR for hypermethylation in the tumor suppressor genes APC, p16INk4A and p14ARF. Additionally, expression of p16INK4A protein in the carcinomas was assessed using immunohistochemistry. Marked differences in hypermethylation were found between esophageal, cardiac and gastric ADC in the APC gene (78% vs. 32% vs. 84%) and in the p16INK4A gene (54% vs. 36% vs. 10%). Hypermethylation of p14ARF was absent from esophageal ADC and present infrequently in cardiac (2%) and gastric ADC (10%). Complete loss of p16INK4A protein expression was detectable in 45% of all tumors and was significantly associated with hypermethylation of the p16INK4A gene (p<0.0001, χ2-test). Our results suggest that hypermethylation of p16INK4A and APC are frequent findings in esophageal, cardiac and gastric ADC. Additionally, the data point to a tumor specific methylation pattern in upper gastrointestinal ADC. © 2004 Wiley-Liss, Inc.