The biochemical properties and immunohistochemical localization of prostaglandin D synthetase were investigated in adult rat skin. The activity of prostaglandin D synthetase, which isomerizes prostaglandin H₂ to prostaglandin D₂, was detected in the 100,000 g supernatant of the homogenate of adult rat skin. Whole skin showed considerable activity (1.9 nmol/min/mg protein), and prostaglandin D₂ was the major prostaglandin among those formed from prostaglandin H₂ in the presence of glutathione. The epidermis, which was separated from whole skin by heating (55°C, 30 s), exhibited about three times higher activity (3.5) than the dermis (1.0). The enzymatic properties of both layers were similar; they were absolutely glutathione-dependent, were inhibited only a few percent by 1 mM 1-chloro-2,4-dinitrobenzene, and were completely absorbed by anti-rat spleen prostaglandin D synthetase antibody. Immunohistochemical studies, using anti-rat spleen prostaglandin D synthetase antibody and the immunoperoxidase method, showed that prostaglandin D synthetase was localized in Langerhans cells (not in keratinocytes) in the epidermis, in macrophages or histiocytes, and also in mast cells in the dermis. Immunoelectron microscopy also supported these findings. These results suggest that prostaglandin D₂ is one of the most important arachidonic acid metabolites and plays a significant role in immunological function in the skin via Langerhans cells and macrophages.