The active principle of a cytosol extract from weanling rat liver representing a putative liver-specific growth factor was partially purified and characterized. “Hepatic stimulator substance” was extracted from the livers of 40- to 60-gm male rats by heat treatment of a homogenate in 35% (w/v) phosphate-buffered saline and subsequent ultracentrifugation. This “heat supernatant” and fractions derived from the subsequent purification steps were tested for growth stimulatory activity in two rat hepatoma cell lines. The undifferentiated, fibroblas-toid-like HTC hepatoma cells did not respond to crude hepatic stimulator substance or any of the partially purified preparations. In contrast, MH1C1 cells, which display some differentiated hepatic functions and epithelial morphology, reacted to hepatic stimulator substance and the purified fractions with a dose-dependent increase of their growth rate in serum-free culture. Although insulin, glucagon and epidermal growth factor showed only a minor effect on MH1C1 cell growth on their own, they were active as permissive or potentiating factors for the expression of the maximal effect of hepatic stimulator substance. Similarly, normal adult rat hepatocytes were only sensitive to hepatic stimulator substance when cultured in the simultaneous presence of epidermal growth factor. Under such conditions, hepatic stimulator substance stimulated hepatocyte entry into the S-phase of the cell cycle 3-fold compared to epidermal growth factor alone. Hepatic stimulator substance did not affect growth of human skin fibroblasts and of the rat intestinal crypt cell line IEC-6. When compared to the supernatant obtained after a 65°C heat step, heating to 95°C increased the biological activity of the extract 2- to 3- fold. MH1C1 cell growth with 50 μg protein per ml of the 95°C heat supernatant in serum-free medium was identical to the growth rate obtained with 10% fetal bovine serum. Precipitation with ethanol reduced the growth-stimulatory activity of hepatic stimulator substance by 60%. The biological activity was concentrated 10- and 100-fold by conventional and high-performance gel filtration, respectively. The biological activity was non-dialyzable and was destroyed by protease treatment. In sodium dodecyl slfate-polyacrylamide gel electrophoresis, the partially purified material showed three bands between M.W. 15,300 and 20,000.