Transforming growth factor .beta. (TGF.beta.), a two-subunit Mr, 25,000 polypeptide, inhibits growth of several epithelial human cancer cell lines and has been proposed as an autocrine growth inhibitor. TGF.beta. activity has been found in conditioned media from some breast cancer cell lines, and TGF.beta. mRNA has been detected in breast cancer cell lines and human breast cancer specimens. In the present study we attempted to characterize the interaction of TGF.beta. with breast cancer cells by examining the biological activity, receptor binding, and secretion of this polypeptide by a panel of estrogen receptor (ER)-positive and ER-negative human breast cancer cell lines. Growth of the four ER-negative lines, MDA231, MDA330, HS578T, and BT20, was exquisitely sensitive to TGF.beta.. Dose-dependent inhibition of monolayer growth, anchorage-independent growth, and of [3H]thymidine incorporation was observed with TGF.beta. concentrations ranging from 1 to 100 pM. Growth of the four ER-positive lines, T47D, ZR75-1, and two MCF7 lines from different laboratories, was unaffected by similar concentrations of TGF.beta.. In receptor-binding studies using 125I-TGF.beta., the four ER-negative lines exhibited specific high affinity TGF.beta. receptors. Binding was a time- and temperature-dependent process. Scatchard analysis of the binding data showed between 2800 and 12900 receptor sites per cell and a Kd between 29 and 160 pM. Epidermal growth factor, insulin, insulin-like growth factors I and II, and transforming growth factor .alpha. did not compete for 125I-TGF.beta. binding. Chemical cross-linking studies with ER-negative breast cancer cells revealed three specific TGF.beta. receptors with molecular weights approximating 400,000, 92,000, and 69,000. The four ER-positive lines had no detectable TGF.beta. binding. Using a radioreceptor assay with A549 cells and a NRK bioassay, TGF.beta. activity was detectable in the conditioned media from the four ER-negative cell lines; media from the ER-positive lines had low levels of TGF.beta. activity. In summary, ER-negative, estrogen-independent cultured human breast cancer cells have receptors for, are inhibited by, and secrete TGF.beta. activity, suggesting the possibility that this polypeptide may function as an autocrine growth inhibitor or as a paracrine growth factor for tumor stromal cells.