Serum-free growth of the human malignant melanoma cell line Hs0294 is associated with production of transforming growth factor-alpha and an autostimulatory melanoma mitogen (melanoma growth-stimulatory activity, MGSA). The transforming activity is characterized by stimulation of anchorage-independent growth of normal rat kidney fibroblasts and competition with 125I-epidermal growth factor for binding to normal rat kidney cells. The second activity, MGSA, stimulates the anchorage-dependent growth of human melanoma cells in serum-free culture medium. When acetic acid extracts of Hs0294 conditioned medium are subjected to Bio-Gel P-30 chromatography followed by reverse-phase high-pressure liquid chromatography, the majority of the transforming growth factor-alpha elutes at 30 +/- 4% acetonitrile, while the major peak of MGSA elutes at 35 +/- 3% acetonitrile. These data indicate that the anchorage-dependent serum-free growth of the Hs0294 human melanoma cell line is apparently dependent upon the autostimulatory melanoma mitogen, MGSA, which is separable from the 125I-epidermal growth factor competing activity produced by these cells.