Normal human bronchial epithelial cells were infected with SV40 virus or an adenovirus 12-SV40 hybrid virus, or transfected via strontium phosphate coprecipitation with plasmids containing the SV40 early region genes. Colonies of morphologically altered cells were isolated and cultured; these cells had extended culture lifespans compared to normal human bronchial epithelial cells. All cultures eventually underwent senescence, with the exception of one which appears to have unlimited proliferative potential. Colonies arising after viral infection were screened for virus production by cocultivation with Vero cells; only viral nonproducer cultures were analyzed further. The cells retained electron microscopic features of epithelial cells, and keratin and SV40 T-antigen were detected by indirect immunofluorescence. All of the cultures were aneuploid with karyotypic abnormalities characteristic of SV40-transformed cells. No tumors formed after s.c. injection of the cells in nude mice. These cells should be useful for studies of multistage bronchial epithelial carcinogenesis.