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Tumor cells produce a variety of hormones and growth factors that are associated with modulation of the growth pattern of malignant cells. Hs294T human malignant melanoma cells produce a monolayer mitogen, melanoma growth stimulatory activity (MGSA), that stimulates the growth of Hs294T cultures in serum-free medium. MGSA has been purified to homogeneity from conditioned medium of Hs294T human malignant melanoma cells using acetic acid extraction of the crude conditioned medium followed by three chromatographic processes, including gel-filtration, heparin-Sepharose, and reverse-phase HPLC. MGSA was eluted from the heparin-Sepharose resin with 0.1-0.3 M NaCl. The binding affinity is similar to that of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) but much less than many endothelial cell-derived growth factors which require significantly higher salt concentrations for elution. These procedures resulted in a final yield of purified MGSA that was significantly greater than yields obtained using previously reported procedures. The homogeneous 16,000 and 13,000 molecular weight moieties obtained by means of these procedures exhibited similar bioactivities (stimulating a 2- to 3-fold increase in Hs294T cell growth) over a 0.06-6 ng concentration range. This bioactivity was progressively inactivated during storage at -80 degrees C. These results indicate that the combination of heparin-Sepharose chromatography and reverse phase-HPLC provides a more efficient means of purification of MGSA.