The long term objective of this proposal is to define mechanistically the regulation of cell proliferation in normal and tumor tissues. Human serum inhibits the proliferation of estrogen-sensitive human breast cancer cells; estrogens overcome this effect in a specific manner. Other sex steroids and growth factors are completely ineffective in reversing this inhibition. The inhibitory effect seems to be due to a heat-stable, protease-sensitive molecule of an apparent molecular weight = 70-90 kDa and an isoelectric print of 4.5-4.8. The specific aim of this application is to further purify this specific inhibitor using a variety of procedures including: hydrophobic interaction and chelating chromatography, preparative isoelectric focusing, gel filtration, 2 dimensional polyacrylamide gel electrophoresis, HPLC/FPLC and mono and polyclonal antibodies. The bioassay to measure the inhibitory properties of the purified fractions makes use of MCF7 human breast tumor cells. The potential clinical relevance of these physiologic inhibitors of the proliferation of breast cancer cells are manifold. They include ways to more accurately prognosticate the response of breast tumors to therapeutic decisions taken when histopathological samples are read; receptors for the plasma-borne inhibitors are anticipated to be a more reliable marker of prognosis than probes used now (estrogen receptor, etc.). Equally important, availability of the inhibitory we are purifying, will allow for testing of hypotheses now being proposed to explain the control of estrogen-sensitive cell proliferation.