We previously reported the induction with N-methyl-N-nitrosourea (MNU) of mouse glandular stomach carcinomas showing a gastric phenotype but variation in histologic appearance, as with human gastric carcinomas. In the present study, we established two cell lines, designated MGT-40 and MGT-93, from MNU-induced mouse glandular stomach carcinomas. These cell lines are keratin-positive and grow as epithelial monolayers in culture, requiring transforming growth factor alpha, epidermal growth factor or insulin/transferrin for optimal growth in addition to 10% fetal bovine serum. Retention of the differentiated phenotype for gastric surface mucous cells has been confirmed by cathepsin E immunohistochemistry and reverse transcriptase-polymerase chain reaction for mouse spasmolytic polypeptide. Neither transplantability in nude mice nor colony formation on soft agar was observed, except in one subline. Chromosome analysis revealed aneuploidy with modal chromosome numbers ranging from 58 to 78 and no specific structural abnormalities. This is the first report of cell lines derived from mouse glandular stomach carcinomas. They should prove useful for studies of the mechanisms of regulation of growth and differentiation.