The Chinese hamster lung fibroblast line, CCl39, displays the properties characteristic of normal secondary cultures of Chinese hamster fibroblasts including: reversible G0 growth arrest (less than 2% labeled nuclei), anchorage dependence, and high serum-growth factor dependence. Injection of CCl39 cells, or anchorage-independent variants, in nude mice leads to tumor formation; however, as we have previously shown (Pérez-Rodriguez et al., 1981b), the resulting tumor clones no longer possess the high serum dependence of injected CCl39 cells. Hormonal growth restraints imposed by the host create an in vivo selection for diminished, or "relaxed," growth factor requirement. To characterize this growth factor "relaxation" further, we have analyzed the mitogenic response of parental CCl39 cells, anchorage-independent clones, and selected tumoral derivatives, to purified growth factors. Two highly purified growth factors, thrombin and insulin, together fulfill the growth factor requirements of CCl39 cells; thrombin (1 U/ml) stimulates the reinitiation of DNA synthesis in G0-arrested CCl39 cells, and insulin (10 micrograms/ml) maximally potentiates this stimulation to the level obtained with 10% fetal calf serum. First, we found no correlation between loss of anchorage dependence and growth factor relaxation. Second, we found that A71 (anchorage independent), a tumoral variant of CCl39 capable of growth arrest, and tumor-derived cells all display an increased sensitivity to thrombin and a diminished requirement for the potentiating action of insulin. Examination of thrombin binding to CCl39, A51 (nontumoral, anchorage independent), and A71 cells revealed that the increased sensitivity to thrombin of A71 cells is not attributable to an alteration in thrombin cell surface receptor number or affinity for thrombin. Rather, under standard conditions of serum or growth factor removal (30 hr), A71 cells maintain a metabolically elevated growth-arrested state, different from that of their nontumoral counterparts. Consequently, much lower concentrations of growth factors are needed to induce a proliferative response in these tumoral cells.