Prostaglandin D₂ (PGD₂), the predominant prostanoid produced by activated mast cells, is implicated in a variety of allergic diseases. PGD₂ exerts its effects through two G-protein coupled receptors, DP and CRTH2. PGD₂ mediates chemotaxis of eosinophils, basophils, and Th2 cells via CRTH2-evoked signaling, suggesting a role for this receptor in allergic disease. To characterize the mouse CRTH2 ortholog (mCRTH2), we amplified the mCRTH2 receptor gene and expressed it in HEK293 cells. Saturation ligand binding isotherms demonstrated high-affinity binding of [³H]PGD₂, with a K_d of 8.8 ± 0.8 nM. Competition binding assays with a panel unlabeled prostanoids demonstrated an order of affinity of 13,14-dihydro-15-keto-PGD₂ (DK-PGD₂) ≥ 15-deoxy-Δ^12,14-PGJ₂ (15d-PGJ₂) ≥ PGD₂ ≥ PGJ₂. [³H]PGD₂ binding was also displaced by the nonsteroidal anti-inflammatory drug indomethacin, with a K_i value of 1.04 ± 0.13 μM. No [³H]PGD₂ displacement was detected using fluribrofen, ibuprofen, or aspirin as competitors at concentrations of up to 30 μM. PGD₂, DK-PGD₂, 15d-PGJ₂, and indomethacin each inhibited intracellular cAMP generation in stable transfectant ER293/mCRTH2 cells through a pertussis toxin (PTX) sensitive pathway, consistent with mCRTH2 coupling to a G_i heterotrimeric G-protein. Activation of mCRTH2 elicited chemotaxis of ER293/mCRTH2 cells in response to PGD₂, indomethacin, and 15d-PGJ₂. mCRTH2-dependent chemotaxis was inhibited by PTX and wortmannin, indicating dependence on G_i and PI 3-kinase signal transduction pathways. These data provide the first pharmacological and functional characterization of the mouse CRTH2 receptor.