Adenocarcinoma of the esophagus (ADCE) with Barrett's mucosa and adenocarcinoma of the cardia (ADCC) are often reported as a single pathological entity. In this study we have used strict anatomical-pathological criteria to distinguish between these two lesions and we have investigated their differences in TP53 mutations, MDM2 gene amplification, and cytokeratin expression. DNA was extracted from the tumor areas of formalin-fixed, paraffin-embedded sections in 26 ADCC and 28 ADCE patients. TP53 mutations were detected by temporal temperature gradient electrophoresis and identified by sequencing. MDM2 amplification was assessed by differential polymerase chain reaction. The expression of cytokeratins 4, 7, and 13 was examined by immunohistochemistry. In ADCC, the male to female ratio was 1.8:1, compared to 27:1 in ADCE. Five ADCC patients had a history of other neoplasms, compared to only one ADCE patient. The two types of tumor differed in the prevalence of TP53 mutations (31% in ADCC and 50% in ADCE) and of MDM2 gene amplification (19% in ADCC and 4% in ADCE), and in the pattern of expression of cytokeratin 7 (positive in 100% of ADCE and in 41% of ADCC) and cytokeratin 13 (positive in 81% of ADCE and in 36.5% of ADCC). ADCE and ADCC differ in their clinical characteristics, in the prevalence of TP53 mutations and MDM2 amplifications, and in the patterns of cytokeratin expression. These results support the notion that ADCC and ADCE are distinct pathological entities.