Microglia are the major myeloid-immune cells of the brain parenchyma. In a steady state, microglia monitor their environment for pathogens or damaged cells. In response to neural injury or inflammation, microglia become competent APCs able to prime CD4+ and CD8+ T lymphocytes. We previously demonstrated that neonatal and adult microglia cross-present exogenous soluble Ags in vitro. However, whether microglia are able to cross-present Ag to naive CD8+ T cells in vivo, within the brain microenvironment, remains undetermined. Here, we have designed an original protocol in order to exclude the involvement in cross-presentation activity of peripheral migrating APCs and of CNS-associated APCs. In C57Bl/6 mice, in which the body but not the head has been properly irradiated, we analyzed the ability of resident microglia to stimulate intracerebrally injected CD8+ T cells in vivo. This study demonstrates for the first time that adult microglia cross-present Ag to naive CD8+ T cells in vivo and that full microglia activation is required to overcome the inhibitory constrains of the brain and to render microglia able to cross-prime naive CD8+ T cells injected in the brain. These observations offer new insights in brain-tumor immunotherapy based on the induction of cytotoxic antitumoral T cells.