Cysteinyl leukotrienes (CysLTs) are potent mediators of inflammation synthesized by the concerted actions of 5‐lipoxygenase (5‐LO), 5‐LO‐activating protein (FLAP), leukotriene C₄ synthase, and additional downstream enzymes, starting with arachidonic acid substrate. CysLTs produced by macrophages, eosinophils, mast cells, and other inflammatory cells activate 3 different high‐affinity CysLT receptors: CysLT₁R, CysLT₂R, and GPR 17. We sought to investigate vascular sites of CysLT₂R expression and the role and mechanism of this receptor in mediating vascular permeability events. Vascular expression of CysLT₂R was investigated by reporter gene expression in a novel CysLT₂R deficient‐LacZ mouse model. CysLT₂R was expressed in small, but not large, vessels in mouse brain, bladder, skin, and cremaster muscle. Intravital, in addition to confocal and electron, microscopy investigations using FIT C‐labeled albumin in cremaster postcapillary venule preparations indicated rapid CysLT‐mediated permeability, which was blocked by application of BAY‐u9773, a dual CysLT₁R/CysLT₂R antagonist or by CysLT₂R deficiency. Endothelial human CysLT₂R overexpression in mice exacerbated vascular leakage even in the absence of exogenous ligand. The enhanced vascular permeability mediated by CysLT₂R takes place via a transendothelial vesicle transport mechanism as opposed to a paracellular route and is controlled via Ca²⁺ signaling. Our results reveal that CysLT₂R can mediate inflammatory reactions in a vascular bed‐specific manner by altering transendothelial vesicle transport‐based vascular permeability.— Moos, M. P. W., Mewburn, J. D., Kan, F. W. K., Ishii, S., Abe, M., Sakimura, K., Noguchi, K., Shimizu, T., Funk, C. D. Cysteinyl leukotriene 2 receptor‐mediated vascular permeability via transendothelial vesicle transport. FASEB J. 22, 4352–4362 (2008)