We have previously established a somatomammotroph cell line (rPC0) derived from normal rat pituitary that secretes GH and PRL. In this study we report that conditioned medium from rPC0 cells (CM-rPC0) exhibited a stimulatory effect on the growth of rat mammary epithelial cells in culture. Fractionation of CM-rPC0 revealed that the growth-promoting activity of CM-rPC0 was associated with a fraction eluting from a diethylaminoethyl-Sephacel column at 0.5 M NaCl. The growth-promoting activity of this fraction was abolished by trypsin and was resistant to dithiothreitol treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the predominant components of the 0.5-M NaCl fraction were proteins migrating at the 14-18K region of the gel. The 0.5-M NaCl fraction did not contain immunodetectable GH or PRL. Further fractionation of CM-rPC0 on a heparin-agarose column showed that the growth-promoting activity eluted at 0.9 M NaCl. The two predominant proteins in this fraction had apparent mol wt of 14.5K and 18.2K. Based on the structural and biological properties, several known hormones and growth factors were excluded from consideration as potential candidates responsible for the growth-promoting effect of the 14-18K proteins. It is postulated that this group of 14-18K proteins contains a new factor(s) that affects the growth of mammary epithelial cells.