The effects of 17β-estradiol and tamoxifen (TAM) on the proliferation of responsive MCF-7 and unresponsive HBC-4 human breast cancer cells were studied in a defined culture medium containing insulin (2 µg/ml), transferrin (2 µg/ml), ethanolamine (2 µm), and selenite (25 nm). MCF-7 cells grew at a population-doubling rate of 2.0 days in serum-free medium and at a rate of 1.7 days in the medium containing 1 mg/ml of the 55–70% ammonium sulfate fraction of bovine serum or 1% dextran-coated charcoal-treated fetal bovine serum. Increasing concentrations of the ammonium sulfate fraction and/or dextran-coated charcoal-treated fetal bovine serum increasingly inhibited the growth of MCF-7 cells but did not inhibit HBC-4 cell growth, indicating that such serum preparations contain some growth inhibitor specific for estradiol-responsive MCF-7 cells. A sufficiently high concentration of exogenous estradiol (100 pm) had the dual action of neutralizing the growth inhibition by the 55–70% ammonium sulfate fraction of bovine serum and dextran-treated charcoal-treated fetal bovine serum and enhancing directly the MCF-7 cell growth maximally 2-fold. Bovine serum albumin fraction V containing globulin remnants also inhibited growth, but globulin-free bovine serum albumin did not. Eliminating growth inhibition by the use of globulin-free bovine serum albumin enabled us to develop an ideal medium for assaying the direct effects of estradiol and TAM on MCF-7 cells. With this medium, we clearly identified (a) a direct mitogenic effect of exogenous estradiol on MCF-7 cells which was initiated at 3 pm and maximized at 0.2 to 10 nm, (b) an acute lethal effect of 1 µm TAM and its prevention by 100 pm estradiol, and (c) a nearly 50-fold increase in the concentration of exogenous estradiol (10 nm) required for maximum growth enhancement in the presence of 1 µm TAM than without TAM (0.2–0.3 nm).