The cooperative action of l7p-estradiol (E2) and polypeptide growth factors in stimulating proliferation of human breast cancer cells in vitro was investigated. To prevent background estrogenic stimulation, only phenol red-free media were used. When cultured in media supplemented with steroidstripped serum in which all polypeptide growth factor activity had been chemically inactivated, MCF7 cells were unable to proliferate and became virtually quiescent. In the additional presence of insulin, epidermal growth factor (EGF), and E2, however, cells proliferated as rapidly as did cells cultured in media supplemented with fetal calf serum. Analysis by DNA flow cytometry showed that in the absence of external growth factors, MCF7 cells became arrested predominantly in t h e Gl/Co phase of the cell cycle. Upon addition of insulin in combination with EGF and E2, however, cells reentered the cell cycle with a high degree of synchrony. When added alone, E2 induced only slight mitogenic effects under these growth factor-defined conditions. In contrast, this steroid induced optimal proliferation in conventional steroidstripped serum, which in itself contained considerable mitogenic activity. Insulin (at 10 pg/ml) was the most potent stimulator of MCF7 cell proliferation under growth factor-defined conditions, resulting in a more than sixfold increase in cell number after 96 hours. Other growth factors such as plateletderived growth factor (PDGF), transforming growth factor (TGFP), and ECF had little effect by themselves and only slightly influenced insulin-induced proliferation. At suboptimal concentrations of insulin (10-100 nglml), however, strong synergism was observed between E2 and insulin in inducing MCF7 proliferation. Using the CG5 cell line, a highly E2-sensitive MCF7 variant, synergism with E, was already observed at 1 nglml insulin. It is concluded that MCF7 cells require insulin (or insulin-like growth factors) for proliferation. At suboptimal insulin concentrations, E2 acts synergistically with insulin, possibly by inducing autocrine production of polypeptide growth factors by these cells.