Exogenous TGF beta inhibits the proliferation of human breast cancer cells in vitro. These cells synthesize and secrete TGF beta into their medium predominantly in a latent form. With neutralizing antibodies against native, biologically active TGF beta (278ab and 282ab), we have examined whether HS578T and MDA-231 breast cancer cells utilize their endogenous TGF beta for growth regulation. Low levels of TGF beta activity were detectable in conditioned medium from confluent monolayers of both cell lines in the absence of acid or protease treatment as measured by radioreceptor assay. When added to subconfluent monolayers of the respective cell line, this untreated conditioned medium inhibited DNA synthesis and cell proliferation. This inhibition was blocked by anti-TGF beta antibodies, whereas nonimmune rabbit IgG had no effect. Similar to exogenous TGF beta 1, this conditioned medium induced a dose-dependent increase in steady-state TGF beta 1 mRNA levels when added to subconfluent HS578T cells; this increase was blocked by the 278ab. Consistent with the above, preincubation of either cell line with anti-TGF beta antibodies increased subsequent specific binding of 125I-TGF beta to cell surface receptors without changing binding affinity. Addition of 278ab to quiescent HS578T or MDA-231 cells induced a dose-dependent increase in [3H]thymidine incorporation. Both antibodies stimulated cell proliferation in serum-free medium and anchorage-independent growth of both cell lines. Finally, incubation of HS578T cells with 278ab under serum-free conditions decreased the basal level of TGF beta 1 message expression. These data indicate that cultured human breast cancer cells utilize endogenously produced TGF beta as an autocrine negative growth regulator.