CBS human colon carcinoma cells are poorly tumorigenic in athymic nude mice, whereas FET colon carcinoma cells are non-tumorigenic. Both cell lines have well differentiated properties in tissue culture. Transforming growth factor α (TGF-α) was ectopically expressed by stable transfection of a TGF- α cDNA under repressible tetracycline control. The TGF-α-transfected cells showed enhanced clonal initiation and shortened lag phase growth in tissue culture without an alteration in doubling time in exponential phase relative to untransfected cells. Furthermore, the TGF-α transfectants showed increased independence from exogenous growth factors in clonal growth assays and induction of DNA synthesis after release from quiescence. Growth factor independence was associated with sustained epidermal growth factor receptor activation in quiescent TGF-α-transfected cells and the requirement of exogenous insulin for stimulation of quiescent cells to reenter the cell cycle. Higher cloning, reduced lag time in tissue, and the acquisition of growth factor independence for DNA synthesis without a change in doubling time of TGF-α-transfected cells indicate that autocrine TGF-α functions by facilitating re-entry into the cell cycle from sub-optimal growth states rather than promoting or controlling the proliferation of actively cycling cells. The modulation of growth regulation by autocrine TGF-α was associated with increased malignant properties as TGF-α transfectants showed increased tumorigenicity in athymic nude mice. The administration of tetracycline reversed the effects of TGF-α expression in these cells both in vivo and in vitro, indicating that the alterations of the biological properties were due to the expression of TGF-α. Since these cells are continuously grown in a completely chemically defined medium without serum supplementation, it was possible to assign the mechanism underlying the generation of growth factor independence to the replacement of a requirement for exogenous insulin in parental cells by autocrine TGF-α.