AMP-activated protein kinase (AMPK), a heterotrimeric serine/threonine kinase, is activated by conditions leading to an increase of the intracellular AMP: ATP ratio. However, how AMPK is regulated under the oxidative stress is completely unknown. In the present study, we examined effects of the oxidative agent H2O2 on AMPK. AMPK was transiently and concentration-dependently activated by H2O2 in NIH-3T3 cells. This activation was tightly associated with an increased AMP:ATP ratio, an electrophoretic mobility shift of AMPK al catalytic subunit, and an increased phosphorylation level of AMPK alpha1 threonine 172, which is a major in vitro phosphorylation site by the upstream AMPK kinase. All of these events were significantly blocked by the pretreatment of 0.5% dimethyl sulfoxide, a potent hydroxyl radical scavenger, indicating that AMPK cascades are highly sensitive to the oxidative stress. Interestingly, a specific tyrosine kinase inhibitor, genistein, further stimulated the H2O2-induced AMPK activity by 70% without altering the AMP:ATP. Taken together, our results suggest that AMP:ATP ratio is the major parameter to which AMPK responds under the oxidative stress, but AMPK may be regulated in part by a tyrosine kinase-dependent pathway, which is independent of the cellular adenosine nucleotides level.