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 α1 catalytic subunit, and an increased phosphorylation level of AMPK α1 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.