This report presents further evidence that the liver is the source of the factor responsible for the initiation and/or stimulation of hepatic regeneration. Initial experiments for the isolation and characterization of the active factor are presented. The factor was isolated from the cytosol of regenerating livers (RLC). After an in vivo exposure to RLC, hepatocytes were pulsed in vitro with 3H-thymidine to measure DNA synthesis. Rat and porcine RLC stimulated DNA synthesis in hepatocytes isolated from growing (nonhepatectomized) livers of weanling rats, or from regenerating livers of adult rats. The ability of porcine RLC to stimulate hepatocyte DNA synthesis demonstrated that the factor responsible was not species-specific. In contrast, normal non-regenerating liver cytosol did not stimulate hepatocyte DNA synthesis. Further experiments also revealed that the factor is heat stable. The activity responsible for the increased DNA synthesis was called hepatocyte proliferation factor (HPF). The assay for detecting HPF activity in the nonhepatectomized recipient will facilitate further characterization and purification of HPF.