Haematopoietic immune cell populations play an important role in the pathogenesis of numerous neurological disorders. To better understand the function of resident mononuclear phagocytes and migrating leukocytes in the central nervous system (CNS), the definition of these populations in healthy individuals is crucial. Therefore, the composition of CNS‐associated leukocytes, isolated from macaque brain tissue, was assessed using multicolor flow cytometry. We established a combination of antibodies directed against nine different antigens that enabled a precise classification of all major immune cell populations in a single tube. Macrophages, dendritic cells (DCs), B and T lymphocytes, and natural killer (NK) cells were differentiated in CNS and peripheral blood. Additionally, microglia cells were detected in the brain. Using this antibody combination also allowed the discrimination of functionally different subsets among the distinct immunocyte populations, for example, CD8 positive cytotoxic T lymphocytes. About 95% of the leukocytes in the brain are microglia cells. Two additional myeloid cell populations, CD14 positive macrophages and CD11c‐positive DCs, were also identified. In contrast to blood, where macrophages are more abundant, DCs outnumbered macrophages in the brain. Among lymphocytes, proportions of CD20 positive B lymphocytes were decreased, and T lymphocytes as well as NK cells were increased in brain compared to blood. Significant changes were also detected for macrophage and T‐cell subpopulations. The nonexclusive expression of certain surface makers on different cell populations demanded a simultaneous classification of all intrathecal immune cells. Knowing their exact composition offers new insights on interaction and regulation in inflammatory processes and will be instrumental to monitor alterations in the course of neurological diseases. © 2011 International Society for Advancement of Cytometry