Serially transplantable rat mammary tumor (RMT) cells are not dependent on exogenous epidermal growth factor (EGF) and insulin-like growth factor-I for continuous growth in serum-free medium. Previously, we found that conditioned medium obtained from these cells contained EGF-like mitogenic activity and stimulated tyrosine phosphorylation of a 185-kDa protein in EGF-dependent mammary epithelial cells. This protein is distinct from the EGF receptor and resembles a 185-kDa tyrosine-phosphorylated protein present in RMT cells themselves. The results of the studies reported here indicate that the tyrosine-phosphorylated pl85 detected in growth factor-independent RMT cells and in human mammary epithelial cells exposed to RMT-conditioned medium was activated erbB-2 protein. Partial purification of the activating factor present in RMT-conditioned medium yielded a heparin-binding growth factor with biochemical properties similar to those of neu differentiation factodheregulin (NDFIHRG). RNA-polymerase chain reaction analysis demonstrated that RMT cells expressed mRNA for NDFIHRG, and western-blot analysis confirmed the presence of the 45-kDa secreted form of NDF/HRG in conditioned medium from the growth factor-independent RMT cells. The biological activity of partially purified rat NDFIHRG was examined and found to be the same as that of the pure growth factor. In addition, we found that RMT-conditioned medium, fractionated on an anion-exchange column and by reverse-phase high-pressure liquid chromatography, contained a potent EGF-like growth factor that was distinct from NDF/HRG. This factor competes with '251-EGF for binding to EGF receptors and has an apparent molecular mass of 6600 Da. This factor copurifies by high-pressure liquid chromotography with pure transforming growth factor-a (TGF-a), and the cells are positive for TGF-a mRNA. Thus, growth factor-independent RMT cells also synthesize and secrete TGF-a. These results indicate that growth factor-independent RMT cells secrete two growth factors with overlapping biological activities and suggest that autocrine loops mediated by these factors are important in the growth factor-independent proliferation of the RMT cells.