Phosphorylation of the histone H2AX (gamma H2AX form) is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that gamma H2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU), we studied the brain occurrence of gamma H2AX in developing, postnatal, adult and senescent (2 years) mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse gamma H2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ) of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, gamma H2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that gamma H2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ) and the granule cell precursors (cerebellum). Conversely, gamma H2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS) degenerative diseases.