A highly sensitive and specific radioimmunoassay for prostaglandin D₂ has been developed and used to determine the basal level and regional distribution of this prostaglandin in rat brain, spinal cord and pituitary. The assay can detect as little as 20 pg of prostaglandin D₂, and the antiserum used shows 20% cross-reactivity to prostaglandin D₁, 0.1% cross-reactivity to 13,14-dihydro-15-ketoprostaglandin D₂ and even lower cross-reactivity to other prostaglandins. Prostaglandin D₂-like immunoreactivity was extracted with ethanol from the rat tissues. The immunoreactivity comigrated with authentic prostaglandin D₂ on silica gel thin layer chromatography, showed the dilution curve parallel to that of the authentic compound, and decreased in amounts by the pretreatment of animals with indomethacin, suggesting that it was prostaglandin D₂ itself. To avoid a postmortem formation of prostaglandins, we sacrificed animals by microwave irradiation at 4.5 kW for 1.2 sec under which conditions both prostaglandin D synthetase and prostaglandin D dehydrogenase were completely inactivated. The amount of prostaglandin D₂ in whole brain measured under these conditions was 3.42±0.59 ng (mean+S.E.M.), and those of prostaglandin E₂ and F_2α measured by the respective radioimmunoassays were 1.32±0.24 and 0.96±0.20 ng, respectively. Prostaglandin D₂ was widely distributed in rat brain, spinal cord and pituitary. The highest concentrations were seen in pineal gland and neurointermediate pituitary followed by anterior pituitary. Lower but significant concentrations were observed in other parts of brain, among which hypothalamus and septum showed the relatively high concentrations.