The unavailability of human cell lines representative of the gastric glandular epithelium while able to form a functional barrier restricts the application of a cell culture approach to the field of gastric epithelial physiology. In the current study, we have characterized new non-transfected clones isolated from gastric carcinoma cell lines known to express functional markers of the human gastric mucosa (J Cell Biochem 2001;81:241). Twenty-one clones exhibiting epithelial-type junctions (renamed HGE cell lines) were isolated from NCI-N87 (ATCC CRL 5822), whereas only squamous cell lines could be generated from other native strains. Of these 21 clones, HGE-17 and HGE-20 formed dense coherent monolayers and displayed true epithelial phenotype. E-cadherin and ZO-1 proteins were consistently localized at the periphery of all cells which also generated transepithelial electrical resistance. Moreover, growth factors known to be trophic for the gastric mucosa were able to stimulate mitogenesis at subconfluence. HGE-17 exhibited a poorly differentiated precursor-like status and responded strongly to EGF/TGFα treatment in restitution assays. HGE-20 cells, on the other hand, exhibited a higher degree of differentiation at the ultrastructural level as well as higher gastric lipase and pepsinogen levels. These latter zymogens were compartmentalized into granules which also contained mucin-6 (MUC6, prezymogenic-like status). Exogenous hormones, i.e., 1 μg/ml hydrocortisone and 5 μM retinoic acid, significantly increased enzyme levels in HGE-20. In conclusion, HGE-17 and HGE-20 represent the first human gastric cell lines with true epithelial characteristics, opening a venue to important applications for the study of re-epithelization, permeability, and regulation of digestive functions in the context of gastric physiology and pathology. © 2005 Wiley-Liss, Inc.