Human squamous carcinoma (COLO-16) cells release factors which specifically stimulate the synthesis of major acute-phase plasma proteins in human and rodent hepatic cells. Anion exchange, hydroxyapatite, lectin, and gel chromatography of conditioned medium of COLO-16 cells result in separation into three distinct forms of hepatocyte-stimulating factors (designated HSF-I, HSF-II, and HSF-III) with apparent molecular weights of 30,000, 50,000 and 70,000, respectively. None of the preparations contains detectable amounts of thymocyte-stimulating activity. Each of the three HSF forms stimulates the accumulation of mRNA for alpha 1-antichymotrypsin in the human hepatoma cell line, HepG2. When the same factors were added to primary cultures of adult rat hepatocytes, the expression of the same set of plasma proteins was modulated as by nonfractionated medium. The hormonally induced accumulation of mRNA for acute phase proteins is qualitatively comparable to that occurring in the liver of inflamed rats. Unlike in human cells, in rat liver cells dexamethasone acts additively and synergistically with HSFs. The only functional difference between the three HSF forms lies in the level of maximal stimulation. HSF-I represents the predominant form produced by normal human keratinocytes and closely resembles in molecular size and isoelectric point the activity produced by activated peripheral blood monocytes while the larger molecular weight forms are more prevalent in human as well as mouse squamous carcinoma cells. The observation that HSFs from different sources elicit essentially the same pleiotropic response in hepatic cells led to the hypothesis that the species-specific reaction of adult liver cells to inflammatory stimuli is pre-programmed and that the function of any HSF is to trigger and tune the execution of this fixed cellular process.