The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) and their class II G protein-coupled receptors VPAC(1), VPAC(2), and PAC(1) play important roles in human physiology. No small molecule modulator has ever been reported for the VIP/PACAP receptors, and there is a lack of specific VPAC(2) antagonists. Via high-throughput screening of 1.67 million compounds, we discovered a single small molecule antagonist of human VPAC(2), compound 1. Compound 1 inhibits VPAC(2)-mediated cAMP accumulation with an IC(50) of 3.8 microM and the ligand-activated beta-arrestin2 binding with an IC(50) of 2.3 microM. Compound 1 acts noncompetitively in Schild analysis. It is a specific VPAC(2) antagonist with no detectable agonist or antagonist activities on VPAC(1) or PAC(1). Compound 2, a close structural analog of compound 1, was also found to be weakly active. To our surprise, compound 1 is completely inactive on the closely related mouse VPAC(2). Chimera experiments indicate that compounds 1 and 2 bind to the seven transmembrane (7TM) region of the receptor as opposed to the N-terminal extracellular domain, where the natural ligand binds. Compound 1, being the first small molecular antagonist that is specific for VPAC(2), and the only VPAC(2) antagonist molecule known to date that allosterically interacts with the 7TM region, will be a valuable tool in further study of VPAC(2) and related receptors. This study also highlights the opportunities and challenges facing small molecule drug discovery for class II peptide G protein-coupled receptors.