Objective To culture normal adult esophageal epithelial cells and establish a normal esophageal epithelial cell line to provide experimental materials for further studies in vitro. Methods Normal esophageal epithelium samples were obtained in sterile phosphate buffered saline supplemented with antibiotics from the normal part of the esophagus of a patient with esophageal carcinoma. Tissue was cleaned of all connective tissues and muscles and rinsed in phosphate buffered saline 5_6 times. The mucosal layer was minced into small pieces and dissociated into a single cell suspension by 025% Dispase and 025% trypsin/002%EDTA. Cells were grown in keratinocyte serumfree media. The cultured cells were identified through their morphological characteristics and immunohistochemical staining. The proliferative capacity of the cultured cells was also examined. Results The cultured cells showed microscopic features of epithelial cells and were positive in keratin and epithelial membrane antigen staining. Eight days after primary culture, the cells displayed a cobblestone morphology reaching 80%_90% confluency and were passaged successfully with 025% trypsin/002%EDTA. The cell cycle analysis showed about 7760% of the cultured cells was in G0/G1 phase and the others in S/G2/M phase. Conclusion\ The culture methods and techniques used in the exp