The rat prostate contains two types of growth factors capable of stimulating DNA synthesis in BALB/3T3 cells. These rat prostatic growth factors (RPGF) were separable by a different affinity for heparin: low affinity type RPGF and high affinity (HiA) type RPGF. About 80% of the RPGF in the cytosol from normal prostates was low affinity type, whereas more than 80% in the cytosol from the Dunning tumors was HiA type. Elution profile of HiA-RPGF showed two peaks of activity eluted from the heparin-Sepharose column, one at 1.3-1.4 M NaCl (HiA1-RPGF) and the other at 1.6-1.7 M NaCl (HiA2-RPGF). HiA2-RPGF could be purified 1100-fold from the Dunning tumor (AT-3 subline) in about 20% recovery by heparin-Sepharose chromatography. The partially purified HiA2-RPGF in the Dunning tumor has a molecular weight of about 19,000 and isoelectric point of about 3.8, and stimulated DNA synthesis at a concentration of about 0.25 nM. The activity was lost by heat treatment at 70 degrees C for 5 min and by acid treatment, whereas it was stimulated by incubating with dithiothreitol. The HiA2-RPGF did not have transforming growth factor activity at a concentration of 250 ng/ml or lower in the presence of epidermal growth factor.