The radiobiology of human tumors suggests that multiple factors are involved in clinical radioresponsiveness. To date, no direct experimental evidence is available to correlate intrinsic cellular radiosensitivity with the steps of malignant transformation. We developed an in vitro multistage model of epithelial neoplasia using human epidermal keratinocytes to examine the effects of malignant transformation on radiation response. These cells were first immortalized as a result of infection with a hybrid virus (adenovirus 12 and simian virus 40) and subsequently transformed either by infection with a second virus (Kirsten murine sarcoma virus) or by treatment with a chemical carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide). We demonstrate that primary human epidermal keratinocytes were radiation resistant (D0 = 2.24 Gy) as compared with human fibroblasts (D0 = 1.45 Gy) and that this resistance was retained in the immortalized as well as the transformed cell lines. These findings present direct experimental evidence that radiation sensitivity of malignant human keratinocytes is an intrinsic property of the precursor cell that may be conserved through the stages of neoplastic transformation.