Some observations have suggested that cells from the central nervous system (CNS) could present exogenous antigens on major histocompatibility complex (MHC) class I molecules to CD8⁺ T cells (a process called cross‐presentation). Microglia are the major myeloid immunocompetent cells of the CNS. When activated, following the injury of the nervous parenchyma, they become fully competent antigen‐presenting cells (APC) that prime CD4⁺ T lymphocytes. We therefore tested the cross‐presentation capacity of murine microglia. We report that a microglial cell line (C8‐B4), neonatal microglia, and interestingly adult microglia cross‐present soluble exogenous antigen (ovalbumin) to a OVA‐specific CD8⁺ T‐cell hybridoma and cross‐prime OVA‐specific naive OT‐1 CD8⁺ T cells. In both these cases, C8‐B4 and neonatal microglia cross‐present OVA as well as peritoneal macrophages. Although cross‐presentation by adult microglia is less efficient, it is increased by GM‐CSF and CpG oligodeoxynucleotide (ODN) stimulation. Using microglial cells either exposed to an inhibitor of proteasome, lactacystin, or purified from TAP^−/− mice, we demonstrate that the microglia cross‐present antigen in proteasome‐ and TAP‐dependant pathways, respectively. Last, microglia purified from adult mice injected intracerebrally with OVA efficiently stimulate OVA‐specific CD8⁺ T cells, thereby showing that microglia take up and process exogenous antigen into MHC class I in vivo. This first demonstration of the cross‐presentation property of microglia offers novel therapeutic approaches to modulate CD8 T‐cell responses in the brain. © 2007 Wiley‐Liss, Inc.