The cysteinyl leukotrienes (cys-LTs) are proinflammatory lipid mediators acting on the type 1 cys-LT receptor (CysLT₁R) to mediate smooth muscle constriction and vascular permeability. GPR17, a G protein-coupled orphan receptor with homology to the P2Y and cys-LT receptors, failed to mediate calcium flux in response to leukotriene (LT) D₄ with stable transfectants. However, in stable cotransfections of 6×His-tagged GPR17 with Myc-tagged CysLT₁R, the robust CysLT₁R-mediated calcium response to LTD₄ was abolished. The membrane expression of the CysLT₁R analyzed by FACS with anti-Myc Ab was not reduced by the cotransfection, yet both LTD₄-elicited ERK phosphorylation and the specific binding of [³H]LTD₄ to microsomal membranes were fully inhibited. CysLT₁R and GPR17 expressed in transfected cells were coimmunoprecipitated and identified by Western blots, and confocal immunofluorescence microscopy revealed that GPR17 and CysLT₁R colocalize on the cell surface of human peripheral blood monocytes. Lentiviral knockdown of GPR17 in mouse bone marrow-derived macrophages (BMMΦs) increased both the membrane expression of CysLT₁R protein by FACS analysis and the LTD₄-elicited calcium flux in a dose-dependent manner as compared with control BMMΦs, indicating a negative regulatory function of GPR17 for CysLT₁R in a primary cell. In IgE-dependent passive cutaneous anaphylaxis, GPR17-deficient mice showed a marked and significant increase in vascular permeability as compared with WT littermates, and this vascular leak was significantly blocked by pretreatment of the mice with the CysLT₁R antagonist, MK-571. Taken together, our findings suggest that GPR17 is a ligand-independent, constitutive negative regulator for the CysLT₁R that suppresses CysLT₁R-mediated function at the cell membrane.