We have isolated a strongly mitogenic, type beta transforming growth factor (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal growth factor (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of 21%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (20 pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on growth-arrested NRK, human lung, and Swiss mouse 3T3 fibroblast monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting factors responsible for the transforming action of culture fluids from FeSV-Fre cells.