Kirsten murine sarcoma virus (KiMSV)-transformed rat-1, normal rat kidney (NRK), and BALB/c 3T3 cells are capable of continual growth in a serum-free medium supplemented with transferrin and insulin but with no exogenous mitogenic growth factors. Cells transformed by a mutant of KiMSV that is temperature sensitive for the maintenance of transformation grow in this medium at the permissive temperature only. At the nonpermissive temperature, growth is dependent upon the presence of serum-free conditioned medium from the transformed cells. Normal rat-1 cells are also dependent upon factors from the transformed cells for growth in this serum-free/mitogen-free medium. The serum-derived growth factors, epidermal growth factor, and fibroblast growth factor have no effect on the transformed cells, although epidermal growth factor can replace transforming growth factors produced by KiMSV-transformed cells for the growth of rat-1 cells. Growth of the transformed cells in serum-free medium at clonal densities is dependent upon the presence of conditioned medium collected from the same cells grown to high densities. These results show that (i) growth in serum-free/mitogen-free medium is a general property of KiMSV-transformed cells and (ii) growth of the transformed cells in this medium is dependent upon the presence of growth factors known to be produced by the cells, and they provide support for the hypothesis that serum-free growth of KiMSV-transformed cells is dependent upon ectopically produced growth factors.