A new human breast cancer cell line (SUM-52PE), originating from a malignant pleural effusion specimen, that can be cultured under serum-free conditions has been isolated. Experiments were conducted to examine the relationship between expression of the erbB family of growth factor receptors and growth regulation in these cells. SUM-52PE cells are epidermal growth factor receptor negative but express single copy levels of erbB-2 protein. Southern blot analysis indicates that the erbB-2 gene is not amplified in these cells. The cells also express mRNA for both erbB-3 and erbB-4. Phosphotyrosine Western blot analysis of membrane protein obtained from SUM-52PE cells indicates the presence of a constitutively tyrosine phosphorylated M(r) 185,000 protein. Immunoprecipitation, using antibodies to erbB-2 or erbB-3, coupled to phosphotyrosine Western blot analysis indicates that both erbB-2 and erbB-3 are constitutively tyrosine phosphorylated in proliferating SUM-52PE cells. Conditioned medium obtained from SUM-52PE cells does not induce tyrosine phosphorylation of p185erbB-2 in a sensitive indicator cell line, suggesting that an erbB-2 activating factor is not secreted by these cells. However, neu differentiation factor/heregulin (NDF/HRG) mRNA is expressed by the cells, and Western blot analysis of SUM-52PE membrane protein revealed the presence of a M(r) 90,000 immunoreactive NDF/HRG protein. Thus, SUM-52PE cells synthesize a membrane bound form of NDF/HRG that may activate erbB-2 and erbB-3 via a juxtacrine mechanism. The addition of exogenous beta-2-NDF/HRG to the culture medium of SUM-52PE cells yields enhanced tyrosine phosphorylation of p185erbB-2/erbB-3 but has only a small stimulatory effect on the proliferation of these cells. By contrast, an erbB-2 monoclonal antibody that binds to the extracellular domain of erbB-2 is potently mitogenic for these cells. SUM-52PE cells were also found, by phosphotyrosine Western blot analysis, to express an inordinately large number of tyrosine phosphoproteins. Direct measurement of phosphotyrosine phosphatase (PTPase) activity in SUM-52PE cell membrane protein revealed 2-3-fold lower levels of PTPase activity compared to other normal and neoplastic breast epithelial cell lines. Thus, SUM-52PE cells exhibit altered growth phenotypes not identified previously in human breast cancer cells. The constitutive activation of erbB-2 and erbB-3 in these cells, coupled with their low, membrane-associated, PTPase activity are likely to play direct roles in driving proliferation of these breast cancer cells.