The role of neuropeptide Y Y2 receptor (Y2R) in human diseases such as obesity, mood disorders, and alcoholism could be better resolved by the use of small-molecule chemical probes that are substantially different from the currently available Y2R antagonist,Presented here are five potent, selective, and publicly available Y2R antagonists identified by a high-throughput screening approach. These compounds belong to four chemical scaffolds that are structurally distinct from the peptidomimetic BIIE0246. In functional assays, IC 50 values between 199 and 4400 nM against the Y2R were measured, with no appreciable activity against the related NPY-Y1 receptor (Y1R). Compounds also displaced radiolabeled peptide YY from the Y2R with high affinity (K i values between 1.55 and 60 nM) while not displacing the same ligand from the Y1R. In contrast to BIIE0246, Schild analysis with NPY suggests that two of the five compounds behave as competitive antagonists. Profiling against a panel of 40 receptors, ion channels, and transporters found in the central nervous system showed that the five Y2R antagonists demonstrate greater selectivity than BIIE0246. Furthermore, the ability of these antagonists to penetrate the blood-brain barrier makes them better suited for pharmacological studies of Y2R function in both the brain and periphery.Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain (Catapano and Manji, 2007) and was originally identified in 1982 based on its structural similarity to peptide YY (70% homology with PYY) and pancreatic polypeptide (50% homology with PP) (Tatemoto, 1982). Each of these 36-residue peptides is processed from a 94-to 95-residue prohormone (Balasubramaniam, 1997). Four related type 1 G protein-coupled receptor (GPCR) family members (Y1, Y2, Y4, and Y5) mediate biological responses to NPY. These receptors couple to the G␣ i signaling pathway and inhibit adenylate cyclase, preventing cAMP production.Although NPY receptors have mainly been of interest to the pharmaceutical industry as therapeutic targets for obesity and feeding disorders, NPY has numerous roles in the body, including modulation of feeding behavior, circadian rhythm,