Evidence is presented indicating that the chemically transformed AKR-MCA and C3H/MCA-58 murine cell lines produce “transforming growth factor(s)” capable of inducing a transformed morphology and the ability to grow in soft agar in nontransformed, anchorage-dependent indicator cells. Serumfree medium conditioned by exposure to the chemically transformed cells was chromatographed on a Bio-Gel P-60 column after dialysis and lyophilization. Using the nontransformed mouse AKR-2B cells as the indicator cells, a peak of soft agar growth-stimulating activity was detected in the molecular weight range of 10,000 to 12,000. The soft agar growth-stimulating activity in pooled fractions from the AKR-MCA cells was shown to be trypsin and dithiothreitol sensitive and relatively heat stable; the activity was not destroyed by heating to 56° for 30 min or to 100° for 3 min. The pooled material also caused stimulation of growth in the soft agar of rat NRK cells and stimulation of DNA synthesis in the AKR-2B cells. The quantity required to give significant competition for binding to the epidermal growth factor receptor was about one order of magnitude greater than that required for stimulation of soft agar growth. Further separation of these polypeptide(s) by carboxymethylcellulose chromatography revealed three apparent peaks of soft agar growth-stimulating activity. Epidermal growth factor receptor-competing activity cochromatographed with the early minor soft agar growth-stimulating peak, whereas the two major peaks of soft agar growth-stimulating activity had no associated detectable competition for epidermal growth factor binding to its receptor. The data indicate that at least a major portion of the transforming growth factors produced by the chemically transformed cells is different from those described previously in murine sarcoma virus-transformed mouse cells and human tumor cells.