—Ligands for peroxisome proliferator–activated receptor γ, such as the thiazolidinedione class of antidiabetic drugs and 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), modulate various processes in atherogenesis. In search of cells that generate prostaglandin D₂ (PGD₂), the metabolic precursor of 15d-PGJ₂, we identified PGD₂ from culture medium of endothelial cells. To study how PGD₂ production is regulated in endothelial cells, we investigated the role of fluid shear stress in the metabolism of PGD₂. Endothelial cells expressed the mRNA for the lipocalin-type PGD₂ synthase (L-PGDS) both in vitro and in vivo. Loading laminar shear stress using a parallel-plate flow chamber markedly enhanced the gene expression of L-PGDS, with the maximal effect being obtained at 15 to 30 dyne/cm². The expression began to increase within 6 hours after loading shear stress and reached the maximal level at 18 to 24 hours. In contrast, shear stress did not alter the expression levels of PGI₂ synthase and thromboxane A₂ synthase. In parallel with the increase in the expression level of L-PGDS, endothelial cells released PGD₂ and 15d-PGJ₂ into culture medium. These results demonstrate that shear stress promotes PGD₂ production by stimulating L-PGDS expression and suggest the possibility that a peroxisome proliferator–activated receptor γ ligand is produced in vascular wall in response to blood flow.