Although del Río Hortega originally reported that leptomeningeal cells are the direct source of ramified microglia in the developing brain, recent views seem not to pay much attention to this notion. In this study, in vitro experiments were conducted to determine whether leptomeninges generate ramified microglia. The leptomeninges of neonatal rats containing Iba1⁺ macrophages were peeled off the brain surface. Leptomeningeal macrophages expressed CD68 and CD163 strongly, but not microglia in the brain parenchyma. Leptomeningeal macrophages expressed epidermal growth factor receptor (EGFR) as revealed by RT-PCR and immunohistochemical staining. Cells obtained from the peeled-off leptomeninges were cultured in a serum-free medium containing EGF for 4 or 6 d resulting in the formation of large cell aggregates, in which many proliferating macrophages were present. In contrast to EGF, macrophage-colony stimulating factor (M-CSF) did not enhance the generation of Iba1⁺ cells from the leptomeningeal culture. The cell aggregates generated ramified Iba1⁺ cells in the presence of fetal calf serum, which express CD68 and CD163 at lower levels than primary microglia isolated from a mixed glial culture. Therefore, the leptomeninge-derived cells resembled parenchymal microglia better than the primary microglia. Furthermore, it was found that cultured leptomeninges gave rise to not only microglia but also other types of cells including NG2+ ramified cells resembling NG2 cells (or oligodendrocytes progenitor cells) in vivo. To see if similar events to these in vitro observations occur in vivo, introduction of lentiviral vector carrying GFP-gene into subdural space is now conducting. This study suggests that microglial progenitors expressing EGFR reside in the leptomeninges and that there is a population of microglia that grow independently of M-CSF, and also suggests the possibility that the leptomeninges function as a source of multiple types of cells in the brain parenchyma.