We have examined the expression of transformed phenotypes and genetic changes associated with the expression of each transformed phenotype after X-ray irradiation. Unirradiated cells grown at a constant growth rate until 8 passages (population doubling number, 15) exhibited little morphological change and ceased to divide thereafter. X-irradiated cells escaped from senescence and showed morphological alteration and anchorage independence after a population doubling number of 20. The acquisition of tumorigenicity in nude mice was observed much later (35 population doublings after irradiation). From cytogenetic analysis, all anchorage-independent clones were consistently found to have trisomy of chromosome 7. Furthermore, cells derived from tumors contained three copies of chromosome 9q in addition to the trisomy of chromosome 7. We have not detected any augmented expression of v-Ha-ras- and v-myc-related oncogenes with RNA dot-blot analysis and could not find activation of any type of oncogenes by NIH3T3 transfection experiments. Our studies demonstrated that X-ray-induced neoplastic transformation is a multistep phenomenon and that the numerical change of specific chromosomes may play an important role in the expression of each transformed phenotype. The results suggest that different endogenous oncogenes, other than the ras gene family and myc oncogene, could be responsible for the progressive nature of neoplastic transformation.