Mouse hepatocyte growth-stimulating factor (mHGSF), which increased markedly in the liver of carbon tetrachloride-treated mice, was purified 275 000-fold with 21% yield from extracts of the injured liver. The purification involves ammonium sulfate precipitation and chromatography on AffiGel Blue, heparin-Sepharose, and S-Sepharose. The purified factor migrated as a major band of 76 000 daltons under nonreducing conditions and two bands of 62 000 and 31 000 daltons under reduced conditions. A dose-response of this growth factor for stimulation of deoxyribonucleic acid synthesis in cultured rat hepatocytes and its maximal effects were similar to those of human hepatocyte growth factor (hHGF), which we previously purified from the plasma of patients with fulminant hepatic failure (E. Gohda et al., J. Clin. Invest., 81, 414-419 (1988)). The effect of mHGSF was additive to the maximal effect of epidermal growth factor and was synergistic with that of insulin or acidic fibroblast growth factor, but was neither additive nor synergistic with the maximal effect of hHGF. mHGSF, like hHGF, was sensitive to heat and trypsin treatments and to reduction by dithiothreitol. This factor did not react with an anti-hHGF antiserum. These results indicate that mHGSF is a hHGF-like factor, but it is immunologically different from hHGF.