High endothelial venules (HEVs) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. Studies with chlorate, a metabolic inhibitor of sulfation, had previously revealed that production of PAPS (3'-phosphoadenosine-5'-phosphosulfate), the high-energy donor of sulfate, is required for sulfation and high-affinity recognition of HEV sialomucins GlyCAM-1 and CD34 by the lymphocyte homing receptor L-selectin. Here, we report the molecular characterization of a novel 2.5 kb human cDNA from MECA-79+ HEV-derived endothelial cells that encodes the target of chlorate, PAPS synthetase, a multifunctional enzyme containing domains for both ATP sulfurylase and adenosine-5'-phosphosulfate kinase. Functional expression of the isolated cDNA in Chinese hamster ovary cells results in high levels of PAPS synthesis, which is abolished by treatment of the transfected cells with chlorate. Northern blot analysis reveals a wide tissue distribution of PAPS synthetase mRNA in the human body, suggesting that human PAPS synthetase may be important for sulfation not only of HEV sialomucins, but also of many other molecules, including mucins such as the P-selectin ligand PSGL-1, proteoglycans, hormones, neurotransmitters, drugs, and xenobiotics.