Hepatic stimulator substance is a liver growth stimulator derived from the hepatocyte cytosol of weanling or regenerating adult rat livers. The present paper reports the almost 9,000-fold purification of hepatic stimulator substance with an approximately 100,000-fold increase in specific growth stimulator activity. Purification steps included heating at 95°C for 15 min, 40% cold ethanol precipitation, passage over Procion Red HE3B, DEAE cellulose and Sephadex G75 columns and gel filtration and reverse-phase fast protein liquid chromatography techniques. As little as 27 ng per ml of the purest material produced a 2-fold stimulation in the standard HTC cell activity assay. Further studies indicate that hepatic stimulator substance is a highly negatively charged protein and that disulfide bonds or a complex tertiary structure are not essential to its activity. Hepatic stimulator substance is stable over a wide range of pH's and temperatures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver stain revealed 1 major band at 12,400 daltons and 1 minor band at 17,500 daltons.