Conditions are described for growing and maintaining the estradiol sensitivity of the human breast cancer cell line ZR-75-1 both in monolayer and suspension cultures. Either newborn calf or fetal calf serum can be used in the culture medium, but an effect of estradiol on growth of the cells was only observed reproducibly if the serum was first treated with dextran-charcoal. Sulfatase treatment of the sera prior to dextran-charcoal treatment did not decrease cell growth in the absence of added estradiol, indicating that estrogen sulfates are unlikely to contribute to cell growth in dextran-charcoal-treated sera. In monolayer cultures, estradiol increased both the growth rate and final saturation density of the cells for each individual plating density tested in a dose-dependent manner with maximal stimulation occurring between 10(-10) and 10(-8) M estradiol. Estradiol also markedly increased the ability of the cells to grow both in suspension and semisolid Methocel cultures. In suspension, the cells grew as tight balls which clustered together to give small organoid-like structures reaching diameters of 4 mm and composed of an outer shell of living cells containing a central cavity of necrotic cells. In the absence of estradiol in both monolayer and suspension, the cells went through a limited and constant number of divisions and then stopped, such that the final cell number was determined by the initial plating density. In the presence of estradiol, this block was removed such that in monolayer cultures the final cell number was independent of plating density. A major loss of estradiol response was found if the cells were grown for 7 to 14 days in the absence of estradiol. This loss of response appeared to be due to a loss of ability to grow rather than to selective cell death within the population.