Background: In the embryo, p63 seems to control development of stratified squamous epithelium. p63 is frequently over-expressed in human squamous cell carcinomas of head and neck. We hypothesized that altered expression of p63 may be involved in the sequential development of esophageal carcinomas induced by duodenal content reflux without carcinogens. Methods: In our study we performed total gastrectomy for rats, followed by esophago-jejunostomy, in order to induce chronic duodenal content reflux esophagitis. The animals were sacrificed sequentially, at the 10th, 20th, 30th and 40th week after surgery and their esophagi were examined. A mouse monoclonal antibody against all p63 isotypes (4A4) (Santa Cruz Biotechnology, Santa Cruz , CA ) was used in this study. Expression and localization of 4A4 was carried out and examined by immuno-histochemical analysis. Results: At 20 weeks post-surgery, squamous hyperplasia, dysplasia and columnar lined epithelium were observed. At 30-40 weeks after surgery, 10/29 rats had developed esophageal cancer ( 2 squamous, 10 adeno and 1 adeno-squamous). The p63 immunohistochemistry staining with 4A4 antibody showed nuclear expression of p63 in all the stages of squamous carcinogenesis including normal squamous basal cells (Fig 1), hyperplasia, dysplasia and carcinoma (Fig 2). In contrast, the cells undergoing adeno carcinogenesis including Barrett’s metaplasia, Barrett’s dysplasia and adenocarcinoma did not stain positively for p63 with 4A4 antibody (Fig 3). Conclusions: As reported in human esophageal cancer, p63 expression may be required to promote the development of neoplastic transformation in cancer cells with squamous differentiation, but not adenocarcinoma.