Two forms of NADP-linked 15-hydroxyprostaglandin dehydrogenase for prostaglandin D 2 were found in the cytosol fraction of human blood platelets. These enzymes were purified by ammonium sulfate fractionation, Blue Sepharose, and Sephadex G-100 column chromatography. The two enzymes differed in molecular weights (65,000 for peak I enzyme and 31,000 for peak II as estimated by gel filtration) and their substrate specificities. The relative rates for reaction with peak I enzyme were: prostaglandin D 2, 100(%); E 2, 14; F 2α, 2; I 2, 29; and B 2, 0; whereas for peak II enzyme, D 2, 100; E 2, 23; F 2α, 61; I 2, 29; and B 2, 131. Prostaglandin D 2 was converted to 15-ketoprostaglandin D 2 and then 13,14-dihydro-15-ketoprostaglandin D 2, which were identified by spectrophotometry and gas chromatography/mass spectrometry, respectively. These metabolites were three orders of magnitude less potent in inhibiting human platelet aggregation than prostaglandin D 2. The results indicated that NADP-linked dehydrogenases participated in the metabolic inactivation of prostaglandin D 2 in the platelets. Furthermore, the dehydrogenase activity for prostaglandin D 2 was high in monkey (0.128 nmol/min · mg at 24 °C) and human platelets (0.066), but was not detectable (less than 0.007) in the rabbit, rat, and chicken. Because prostaglandin D 2, which was demonstrated by several authors to be synthesized in platelet-rich plasma during platelet aggregation, exhibited significant antiaggregatory activity only in human and monkey platelets, these prostaglandin dehydrogenases appear to play a physiological role in the circulatory system.