Prostanoid receptors belong to the family of G protein-coupled receptors with seven transmembrane domains. By taking advantage of nucleotide sequence homology among the prostanoid receptors, we have isolated and identified a cDNA fragment and its gene encoding a mouse prostaglandin (PG) D receptor by reverse transcription polymerase chain reaction and gene cloning. This gene codes for a polypeptide of 357 amino acids, with a calculated molecular weight of 40,012. The deduced amino acid sequence has a high degree of similarity with the mouse PGI receptor and the EP2 subtype of the PGE receptor, which together form a subgroup of the prostanoid receptors. Chinese hamster ovary cells stably expressing the gene showed a single class of binding sites for [#H]PGD2 with a Kd of 40 nM. This binding was displaced by unlabeled ligands in the following order: PGD2 > BW 245C (a PGD agonist) > BW A868C (a PGD antagonist) > STA2 (a thromboxane A2 agonist). PGE2, PGF2 alpha, and iloprost showed little displacement activity at concentrations up to 10 microM. PGD2 and BW 245C also increased cAMP levels in Chinese hamster ovary cells expressing the receptor, in a concentration-dependent manner. BW A868C showed a partial agonist activity in the cAMP assay. Northern blotting analysis with mouse poly(A)+ RNA identified a major mRNA species of 3.5 kb that was most abundantly expressed in the ileum, followed by lung, stomach, and uterus.