We applied high-resolution laser-scanning microscopy, electron microscopy, and non-radioactive in situ hybridization histochemistry to determine the cellular and intracellular localization of lipocalin-type prostaglandin D synthase, the major brain-derived protein component of cerebrospinal fluid, and its mRNA in leptomeninges, choroid plexus, and parenchyma of the adult rat brain. Both immunoreactivity and mRNA for prostaglandin D synthase were located in arachnoid barrier cells, arachnoid trabecular cells, and arachnoid pia mater cells. Furthermore, meningeal macrophages and perivascular microglial cells, identified by use of ED2 antibody, were immunopositive for prostaglandin D synthase. In the arachnoid trabecular cells, the immunoreactivity for prostaglandin D synthase was located in the nuclear envelope, Golgi apparatus, and secretory vesicles, indicating the active production and secretion of prostaglandin D synthase. In the meningeal macrophages, prostaglandin D synthase was not found around the nucleus but in lysosomes in the cytoplasm, pointing to an uptake of the protein from the cerebrospinal fluid. Furthermore, the existence of meningeal cyclooxygenase (COX) -1 and COX-2 was investigated by Western blot, Northern blot, and reverse transcriptase-polymerase chain reaction (RT-PCR), and the colocalization of COX-2 and prostaglandin D synthase was demonstrated in virtually all cells of the leptomeninges, choroid plexus epithelial cells, and perivascular microglial cells, suggesting that these cells synthesize prostaglandin D(2) actively. Alternatively, oligodendrocytes showed prostaglandin D synthase immunoreactivity without detectable COX-2. The localization of lipocalin-type prostaglandin D synthase in meningeal cells and its colocalization with COX-2 provide evidence for its function as a prostaglandin D(2)-producing enzyme.