Roles of various growth factors in growth of human osteosarcoma cells which can grow in protein-free medium. The human osteosarcoma cell line (OST-1-PF) can grow in protein-free Coon's modified Ham's F12 medium. Growth of the cells in protein-free medium was partially density-dependent and partially depressed by medium change. An extract and conditioned medium of OST-1-PF cells contained high mitogenic activity for BALB/c3T3 cells. The growth factor in the cells was purified and identified as a basic fibroblast growth factor (bFGF)--like factor on the basis of its elution profile on heparin-affinity chromatography and the result of immunoblotting. An unidentified factor in a conditioned medium eliciting most of the DNA synthesis-stimulating activity showed a weak affinity for heparin. Various additions, including serum and growth factors, stimulated the growth of OST-1-PF cells in protein-free medium. Of these factors, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), acidic fibroblast growth factor (aFGF) and bFGF were the most potent mitogens. High-affinity receptors of EGF and FGF were found on the surface of these cells. These results indicate that autonomous growth of OST-1-PF cells in protein-free medium is mainly controlled by an intracellular mechanism.