The Ataxia-Telangiectasia mutated (ATM) kinase is regarded as the major regulator of the cellular response to DNA double strand breaks (DSBs). In response to DSBs, ATM dimers dissociate into active monomers in a process promoted by the Mre11-Rad50-Nbs1 (MRN) complex. ATM can also be activated by oxidative stress directly in the form of exposure to H₂O₂. The active ATM in this case is a disulfide-crosslinked dimer containing 2 or more disulfide bonds. Mutation of a critical cysteine residue in the FATC domain involved in disulfide bond formation specifically blocks ATM activation by oxidative stress. Here we show that ATM activation by DSBs is inhibited in the presence of H₂O₂ because oxidationblocks the ability of MRN to bind to DNA. However, ATM activation via direct oxidation by H₂O₂ complements the loss of MRN/DSB-dependent activation and contributes significantly to the overall level of ATM activity in the presence of both DSBs and oxidative stress.