Hepatic stimulator substance (HSS) protects liver cells from various toxins. However, the mechanism by which HSS protects hepatocytes remains unclear. In this study, we report that the HSS gene, after transfection into BEL-7402 hepatocma cells, is stably expressed in the mitochondria. Hydrogen peroxide (H(2)O(2))-induced cell apoptosis in the HSS-transfected cells is reduced, as shown by morphologic analysis. In the HSS-transfected cells, disruption of mitochondrial transmembrane potential (MTP) and cytochrome c leakage are reduced. The anti-apoptotic gene Bcl-2 is also highly expressed. In addition, ATP levels in the HSS-transfected cells are maintained. In conclusion, in hepatoma cells, HSS gene expression protects cells against H(2)O(2) injury, and this effect is likely to be associated with preservation of mitochondria.