Glutathione-Independent prostaglandin D synthase, identical to beta-trace, (a major CSF protein), is localized in the CNS. This enzyme, lipocalin-type prostaglandin D synthase, is a member of the lipocalin family of secretory proteins that transport small lipophilic substances. This enzyme's activity in adult rat retina was enriched sixfold in retinal pigment epithelium (RPE) and even more in interphotoreceptor matrix (IPM), all higher than brain. Western blots with anti-lipocalin-type prostaglandin D synthase showed three distinct immunoreactive bands. In the retinal cytosolic fraction, only one band was observed (M(r) 25,000); in IPM, the larger component occurred (M(r), 26,000). The RPE membrane-bound fraction showed two bands (M(r) 20,000 and 23,000), indicating synthesis, and the cytosolic fraction contained two bands (M(r) 23,000 and 26,000), indicating modification for release into IPM. At least two glycosylation sites occurred on the prostaglandin D synthase moiety, explaining the three immunoreactive bands in Western blots. Immunohistochemistry with polyclonal antibodies against this lipocalin-type enzyme showed intense localization in RPE, but less in photoreceptor outer and inner segments. In situ hybridization showed mRNA specifically expressed in RPE. Thus, lipocalin-type prostaglandin D synthase is predominantly expressed in RPE and actively accumulated in IPM. This may demonstrate gene sharing because, while catalyzing prostaglandin D2 synthesis, it may perform an additional, unrelated role in IPM. This enzyme is secreted from the RPE into IPM from which it is then taken up by photoreceptors. However, the nature of its ligand(s) is not known; they may be retinoids and/or docosahexanoic acid.