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Extracts of conditioned medium (CM) from Hs0294 human malignant melanoma cells stimulate [3H]thymidine incorporation and an increase in cell number in serum-depleted Hs0294 cells. This activity is acid and heat stable, nonproteolytic, protease sensitive, contains disulfide bonds and elutes broadly from a Bio-Gel P-30 column with an approximate molecular weight range of 6,000 to 25,000. Hs0294 CM also stimulates [3H]thymidine incorporation in nonmalignant human nevus cells and normal rat kidney fibroblast cells but not in human fibroblasts. There was only limited competition with 125I-epidermal growth factor in binding assays. Hs0294 CM extracts stimulate anchorage-independent growth in normal rat kidney fibroblast cells in soft agar but not in Hs0294 cells, nevus cells, or human fibroblasts. This second activity elutes from the Bio-Gel P-30 column in two positions with apparent molecular weights of 27,000 and 11,000.