Three normal human fibroblast strains were immortalized by treatment with either 4-nitroquinoline 1-oxide (SUSM-1 and OUMS-24F lines) or 60Co gamma rays (KMST-6 line). Immortalization of normal human cells with these agents required repeated treatments. Before immortalization, most of the cells treated with these mutagenic agents showed more or less chromosome aberrations, but immortalized cells appeared very rarely. This suggests that several mutational events are involved in the immortalization process itself. The immortalized cells showed polygonal or epithelial-like morphology, prominent numerical and structural abnormalities of chromosomes, requirement of serum growth factors for their growth, and no tumorigenicity. The immortalized KMST-6 and OUMS-24F were further transformed into neoplastic ones by the ras oncogene, but not by transfection with the mutant p53 gene. Normal human fibroblasts which are mortal were not immortalized nor neoplastically transformed with the ras oncogene or the mutant p53. These findings strongly indicate that the immortalization of human cells is prerequisite for neoplastic transformation of the cells.