The factors involved in estradiol-17 beta induced growth stimulation of MCF-7 human breast cancer cells have been examined. Wild type MCF-7 cells (and clone E3) were shown to undergo slow growth in phenol-red-free medium containing specific calf sera. The E3 clone was used to document a mean 6-day growth stimulation of 3.35-fold (doubling time = 33 +/- 3 h) in cultures supplemented with 10(-11) M estradiol-17 beta. The serum batch utilized in the culture medium is most important in acquiring significant growth stimulation of MCF-7 cells by estradiol-17 beta. Regardless of the absence of phenol-red, only selected sera (2 out of 14 tested) supported minimal growth of MCF-7 cells in the absence of added estradiol 17 beta (doubling time = 55 +/- 11 h). When a calf-serum-supplemented culture failed to display a complete growth response to estradiol-17 beta, it was due to the rapid growth of the cells in the control (minus estradiol-17 beta) flasks. Sera that promoted shorter doubling times for MCF-7 cells cultured in the absence of estradiol-17 beta were rendered less supportive of growth if treated with dextran-coated charcoal or when cultures were supplemented with the estrogen antagonist ICI 164,384 (10(-7) M). Pooled extracts of these sera were shown to contain stimulatory levels of estradiol-17 beta. Dextran-coated charcoal treatment of sera removed or deactivated factors (other than estradiol-17 beta) which were not only required for the growth of MCF-7 cells, but were necessary for estrogen-stimulated growth. Varying the serum-containing medium, buffer, and nutrient mix or the addition of insulin has no effect on the growth response of these cells to estradiol-17 beta. These investigations document the culture conditions required to produce a maximal and consistent proliferative effect of E2 on MCF-7 cells without exposing the serum constituent to damaging chemical or absorbent agents.