Non-tumorigenic SV40-immortalized human cells may be transformed to tumorigenicity by activated oncogenes, but the molecular genetics of this process are still poorly understood. We describe here 4SV40-transformed bronchial epithelial (BE) cell lines that became immortalized after a period of crisis, and then transfection of 6 BE lines or sub-lines with an activated c-Ha-ras (EJ-ras) oncogene. pSV2neo-transfected cells did not form any tumors in athymic nude mice. Even though each of the EJ-ras-transfected lines was shown to be expressing the mutant ras gene, only one cell line, BEAS-2B, and 2 of its sub-lines were tumorigenic after transfection. We conclude that immortalization is not sufficient for BE cells to be transformed by the EJ-ras oncogene. Thus there are at least 2 unknown genetic events in this in vitro model of carcinogenesis: escape from crisis (immortalization), and development of ability to cooperate with activated ras in tumorigenic transformation. We found no evidence that either immortalization or ability to complement ras is related to abnormalities of the SV40 T antigens, of p110RB or of p53.