The vasoactive cyclic 11-amino acid peptide urotensin II (U-II) has recently been discovered as the endogenous ligand of the orphan G-protein-coupled receptor GPR14. As U-II might be involved in the regulation of cardiovascular homeostasis and pathology, a nonpeptidic GPR14/U-II antagonist is of considerable basic and therapeutic interest. We have performed structure-activity relationship studies on U-II by investigating 25 peptide analogues to mobilize intracellular calcium in GPR14-transfected CHO cells, demonstrating that only the side chains of the residues Trp-7, Lys-8, and Tyr-9 are required for receptor recognition and activation. The solution structure of U-II derived by nuclear magnetic resonance has served as a structural template for a three-dimensional three point pharmacophore query for the virtual screening of the Aventis compound repository for nonpeptidic U-II receptor antagonists. Highly active lead compounds of six different scaffold classes could be identified, antagonizing the biological activity of U-II in vitro. The most potent compound identified by the virtual screening approach, 1-(3-carbamimidoyl-benzyl)-4-methyl-1H-indole-2-carboxylic acid (naphthalen-1-ylmethyl)amide, reveals an IC(50) of 400 nM in a functional fluorometric imaging plate reader assay and constitutes a promising lead.