We performed this study to determine whether human mesothelial cells are capable of undergoing neoplastic change in vitro and to observe their interaction with the activated c-Ha-ras (HRAS1) oncogene EJ-ras, which has a role in the development of many malignant human tumors. Mesothelial cells are presumed to be the progenitor cells of malignant mesothelioma, a cancer strongly correlated with asbestos exposure. Previously, we established a non-tumorigenic cell line, MeT-5A, from normal human mesothelial cells after transfection with a plasmid containing the simian virus 40 (SV40) early-region genes. In the present study, we performed transfection of a plasmid containing the EJ-ras gene and the neomycin-resistance gene into these cells and selected a population resistant to G418, a neomycin analogue. Cells from this cell line formed rapidly growing sc tumors in NIH Swiss athymic nude mice, but untransfected with the vector DNA and selected for G418 resistance formed no tumors. The tumors formed by EJ-ras-transfected cells were established in vitro, and cells from these tumor cell lines exhibited a characteristic altered morphology. The cells had the same isoenzyme phenotype as the parent cells, and they expressed the mutant EJ-ras p21 protein. This first demonstration of malignant transformation of human mesothelial cells in vitro may permit molecular analysis of mesothelial carcinogenesis.