The nuclear foci of phosphorylated histone H2AX (γH2AX) are frequently used as a 
marker for DNA double-strand breaks (DSBs) following ionizing radiation (IR). 
However, recent studies reported that γH2AX foci do not necessarily correlate 
with DSBs under other conditions. We showed that γH2AX foci induced by oxidative 
stress in hydrogen peroxide (H2O2)-treated cells displayed several different 
features from those induced by IR. The magnitude of γH2AX induction was 
heterogeneous among H2O2-treated cells. Some cells expressed small discrete 
γH2AX foci, whereas others expressed a gross γH2AX signal that was distributed 
throughout the nucleus. Oxidative stress-induced γH2AX was eliminated in DSB 
repair-deficient mutant cells as efficiently as in wild-type cells and was not 
necessarily accompanied by phosphorylated ataxia telangiectasia mutated (ATM) or 
53BP1 foci. Analyses using specific inhibitors showed that ATM- and Rad3-related 
(ATR), rather than ATM, was the prominent kinase mediating the oxidative stress 
response. These results suggest that a major fraction of γH2AX induced by 
oxidative stress is not associated with DSBs. Single-stranded DNA arisen from 
stalled replication forks can cause the ATR-mediated induction of γH2AX. 
However, oxidative stress appeared to induce γH2AX in both S- and non-S-phase 
cells. These results suggest that there may be another pathway leading to the 
ATR-mediated induction of γH2AX in non-S-phase cells without DSBs.