In vitro transformation of brain cells cultured from various age rats by human adenovirus type 12 (Ad-12) was studied. In cultured brain cells, five cell types could be identified light microscopically. In astrocytes, Ac-2 and Ac-1, astroprotein was demonstrated by immunofluorescence technique. In the former, intracytoplasmic microfilaments were recognized by electron microscopy. Undifferentiated cells (Ud), ependymoglioblasts (EGb) and Ac-1, grew prominently in cultures from young animals or of early passage levels. Ac-2 and fibroblasts were prominent in cultures from aged animals or of late passage levels. Transformed foci appeared within about 20 days after inoculation of Ad-12 and often located in close association with the sheets of Ac-1. Transformed cells morphologically resembled EGb or transitional cells from EGb to Ac-1. Transformation rate was high in cultures from young animals or of early passage levels. Subcutaneous transplantation of these cells into newborn rats procued tumors whose histology was similar to Ad-12-induced rat brain tumor. T-Ag were detected both in transformed and transplanted tumor cells. It was concluded, that the target cells of Ad-12 in vitro correspond with immature brain cells (EGb and/or transitional cells from EGb to Ac-1 cells.