The mechanisms of signal transduction of c-jun induction by hydrogen peroxide are elucidated in NIH3T3 cells by using trapping agents of hydroxyl free radical or inhibitors of various protein kinases. Pre-treatment of the cell with hydroxyl radical scavenger dimethyl sulfoxide (DMSO) abolishes the H2O2-induced c-jun expression. Hydroxyl radical generation can be detected and quantified in cells treated sequentially with DMSO and H2O2 for 30 min respectively by methane sulfinic acid (MSA) production, especially that from particulate fraction. Induction of c-jun by H2O2 is also dramatically reduced by pretreating the cells with biological antioxidant vit. E. Protein tyrosine kinase activity of membrane fraction is induced by H2O2 within 5 to 10 min, which can be prevented by DMSO pre-treatment. Inhibitor of non-receptor type tyrosine kinase, herbimycin A, has inhibitory effect on H2O2-induced c-jun expression while the inhibitor of receptor type tyrosine kinase, tyrphostin 23 or inhibitor of cyclic AMP dependent protein kinase, KT 5720, has not. TPA pre-treatment that depletes protein kinase C (PKC) has no influence on the c-jun induction by H2O2. Our results suggest that the highly reactive species HO is generated after H2O2 enter cells and mediate the signal responses of H2O2 including c-jun induction and the activation of p60src tyrosine kinase might be one of the molecular events associated with the c-jun induction pathway.