Reactive oxygen species (ROS) are important for intracellular signaling mechanisms regulating many cellular processes. Manganese superoxide dismutase (MnSOD) may regulate cell growth by changing the level of intracellular ROS. In our study, we investigated the effect of ROS on 7721 human hepatoma cell proliferation. Treatment with H2O2 (1-10 microM) or transfection with antisense MnSOD cDNA constructs significantly increased the cell proliferation. Recently, the mitogen-activated protein kinases (MAPK) and the protein kinase B (PKB) were proposed to be involved in cell growth. Accordingly, we assessed the ability of ROS to activate MAPK and PKB. PKB and extracellular signal-regulated kinase (ERK) were both rapidly and transiently activated by 10 microM H2O2, but the activities of p38 MAPK and JNK were not changed. ROS-induced PKB activation was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002, suggesting that PI3-K is an upstream mediator of PKB activation in 7721 cells. Transfection with sense PKB cDNA promoted c-fos and c-jun expression in 7721 cells, suggesting that ROS may regulate c-fos and c-jun expression via the PKB pathway. Furthermore we found that exogenous H2O2 could stimulate the proliferation of PKB-AS7721 cells transfected with antisense PKB cDNA, which was partly dependent on JNK activation, suggesting that H2O2 stimulated hepatoma cell proliferation via cross-talk between the PI3-K/PKB and the JNK signaling pathways. However, insulin could stimulate 7721 cell proliferation, which is independent of cross-talk between PI3-K/PKB and JNK pathways. In addition, H2O2 did not induce the cross-talk between the PI3-K/PKB and the JNK pathways in normal liver cells. Taken together, we found that ROS regulate hepatoma cell growth via specific signaling pathways (cross-talk between PI3-K/PKB and JNK pathway) which may provide a novel clue to elucidate the mechanism of hepatoma carcinogenesis.