Villin is a cytoskeletal protein that is involved in the formation of brush-border microvilli in normal small intestine and colon epithelium. This protein is present in Barrett's metaplasia but is reported not to be expressed in Barrett's adenocarcinoma. In this study, we analyzed villin protein expression in Barrett's metaplasia and in both Barrett's adenocarcinomas and tumors of the gastric cardia. Immunohistochemical analysis was used to evaluate the expression and cellular localization of the villin protein in 21 cases of Barrett's metaplasia, 30 cases of Barrett's adenocarcinoma, 16 cases of gastric cardia adenocarcinoma, and eight cases of adenocarcinoma of the distal esophagus. Southern, northern, and western blot analyses were used to evaluate the potential mechanisms for regulation of villin protein expression. Villin protein expression was observed in 21 of 21 cases (100%) of intestinal-type Barrett's metaplasia and in 28 of 30 cases (93%) of Barrett's adenocarcinoma and was thus highly expressed in these tumors. Northern blot analysis demonstrated villin mRNA (3.5 and 2.7 kb) in both villin-positive Barrett's metaplasia and adenocarcinomas. Western blot analysis with the antibody used for immunohistochemical analysis confirmed the presence of a single villin protein band of 95 kDa. Abundant villin expression also was present in both adenocarcinoma of the gastric cardia (13 of 16 cases; 81%) and distal esophageal adenocarcinomas of unknown origin (six of eight cases; 75%). The intestinal brushborder enzyme sucrase isomaltase was found to be present in only 22 of 46 cases (48%) of the adenocarcinomas that expressed villin. We concluded that the protein villin is highly expressed in Barrett's adenocarcinomas and is well maintained in these and other esophageal tumors.