In this study, we investigated the mRNA and protein expression of S100A2 and S100A4 in adenocarcinomas of the stomach and esophagus. Real-time reverse transcription-polymerase reaction analysis on 72 tumors revealed frequent overexpression of S100A2 and S100A4 in Barrett's adenocarcinomas (BAs) (P < .01). Immunohistochemical analysis on tumor tissue microarrays that contained 187 tumors showed absent to weak staining for S100A2 in all normal gastric mucosa samples, whereas normal esophageal mucosa samples demonstrated moderate to strong nuclear staining. Contrary to the nuclear expression of S100A2 in normal esophageal mucosa, two thirds of Barrett's dysplasia and BAs that overexpressed S100A2 demonstrated stronger cytosolic staining than nuclear staining (P < .001). Overexpression of S100A2 protein was more frequently seen in well-differentiated tumors than in others (P = .02). Moderate to strong staining of S100A4 was detected in two thirds of tumors and was frequently observed in the presence of Barrett's esophagus (P = .02). Similar to S100A2, the expression of S100A4 was predominantly cytosolic in two thirds of the tumors (P = .001). There was a significant correlation between S100A4 overexpression and lymph node metastasis (N(2)-N(4)) (P = .027). These results demonstrate frequent cytosolic overexpression of S100A2 and S100A4 in BAs. Further studies are ongoing to understand the biological significance of these S100A proteins in Barrett's tumorigenesis.