AbstractN‐ethyl‐N‐nitrosourea (ENU)‐induced gliomas, animal models of human gliomas, are most frequently oligodendrocytic, while human gliomas tend to be astrocytic. To facilitate a detailed study of human glial carcinogenesis, we developed an in vitro system using type 1 astrocyte transformation with ENU. Type 1 astrocytes from fetal Wistar rat brain were treated by a single dose of ENU. Transformed colonies appeared 50 days after exposure to single doses of ENU greater than 150 μg/mL. Cloned cells from these colonies retained the immunohistochemical characteristics of type 1 astrocytes. They showed rapid growth and high saturation densities, colony formation in low (2%) serum medium and gave rise to tumors when injected into nude mice. When p53expression was studied at each passage, a single cell positive for mutant p53protein emerged 40 days after ENU treatment. In the next 1–3 passages, the mutant p53positive cell formed piled‐up colonies and exhibited dominant growth. Northern blot analysis showed markedly increased accumulations of p53mRNA in transformed cells. This in vitro transformation system of type 1 astrocytes provides a valuable tool for further investigations of astrocyte carcinogenesis. © 1995 Wi