The phosphorylated form of histone H2A.X (γ-H2AX) is a well documented early, sensitive, and selective marker of DNA double-strand breaks (DSBs). Previously, we found that excessive glutamatergic activity increased γ-H2AX in neurons in vitro. Here, we evaluated γ-H2AX formation in the adult rat brain following neuronal excitation evoked by seizure activity in vivo. We found that brief, repeated electroconvulsive shock (ECS)-induced seizures (three individual seizures within 60 min) did not trigger an increase γ-H2AX immunostaining. In contrast, a cluster of 5-7 individual seizures evoked by kainic acid (KA) rapidly (within 30 min) induced γ-H2AX in multiple neuronal populations in hippocampus and entorhinal cortex. This duration of seizure activity is well below threshold for induction of neuronal cell death, indicating that the γ-H2AX increase occurs in response to sublethal insults. Moreover, an increase in γ-H2AX was seen in dentate granule cells, which are resistant to cell death caused by KA-evoked seizures. With as little as a 5 min duration of status epilepticus (SE), γ-H2AX increased in CA1, CA3, and entorhinal cortex to a greater extent than that observed after the clusters of individual seizures, with still greater increases after 120 min of SE. Our findings provide the first direct demonstration that DNA DSB damage occurs in vivo in the brain following seizures. Furthermore, we found that the γ-H2AX increase caused by 120 min of SE was prevented by neuroprotective preconditioning with ECS-evoked seizures. This demonstrates that DNA DSB damage is an especially sensitive indicator of neuronal endangerment and that it is responsive to neuroprotective intervention.