Barrett's esophagus is a condition in which the normal stratified squamous epithelium is replaced by metaplastic columnar epithelium that predisposes to the development of esophageal adenocarcinoma. Neoplastic progression in Barrett's esophagus occurs by a multistep process associated with genomic instability and the development of aneuploid cell populations. p53 protein overexpression and allelic deletions on chromosome 17p have been shown to be present in some Barrett's adenocarcinomas, but the stage in neoplastic progression at which p53 protein overexpression develops has not been investigated. To determine the stages in neoplastic progression at which p53 protein overexpression could be detected, biopsy specimens from patients with Barrett's esophagus at all stages of histological progression from Barrett's metaplasia negative for dysplasia to esophageal adenocarcinoma were investigated using a multiparameter flow-cytometric assay. p53 protein overexpression was found in 1 of 21 patients (5%) with Barrett's metaplasia negative for dysplasia, 2 of 13 patients (15%) with Barrett's metaplasia with abnormalities in the indefinite/low-grade dysplasia range, 5 of 11 patients (45%) with high-grade dysplasia, and 8 of 15 patients (53%) with Barrett's adenocarcinoma (P less than 0.01). p53 protein overexpression was found in 9% of patients with Barrett's esophagus who had neither high-grade dysplasia nor adenocarcinoma. Whether or not patients whose biopsy specimens show p53 protein overexpression are at increased risk for progression to adenocarcinoma can be determined by prospective endoscopic surveillance.