Melanoma growth stimulatory activity (MGSA) is an endogenous growth factor produced by human malignant melanoma cells. When purified from conditioned medium from the Hs0294 human melanoma cell line, MGSA has been shown to be most reproducibly associated with a 16 kDa acid and heat stable moiety which is sensitive to trypsin and dithiothreitol. Monoclonal antibodies to MGSA have been produced. This report describes the use of FB2AH7 monoclonal antibody to prepare an immunoaffinity resin which binds the MGSA produced by Hs0294 cells. This resin was used to purify the MGSA antigen from Hs0294 human melanoma extracts and medium conditioned by the Hs0294 melanoma cell line. The immunoaffinity eluant obtained from both sources produced a significant stimulation in [3H]thymidine incorporation into DNA in Hs0294 cells and yielded similar profiles when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon elution of the material from the polycrylamide gels, a 16 kDa moiety was found to be more reproducibly active in the MGSA [3H]thymidine bioassay and enzyme-linked immunoadsorbent assay. The bioactivity of the 16 kDa material represents a 235,000-fold purification of MGSA.