Endogenous ligands acting on a human P2Y(12) receptor, one of the G-protein coupled receptors, were searched by in silico screening against our own database, which contains more than 500 animal metabolites. The in silico screening using the docking software AutoDock resulted in selection of cysteinylleukotrienes (CysLTs) and 5-phosphoribosyl I-pyrophosphate (PRPP), with high free energy changes, in addition to the known P2Y(12) ligands such as 2MeSADP and ADP. These candidates were subjected to an in vitro Ca2+ assay using the CHO cells stably expressing P2Y(12)-G(16)alpha fusion proteins. We found that CysLTE4 and PRPP acted on the P2Y(12) receptor as agonists with the EC50 values of 1.3 and 7.8 nM, respectively. Furthermore, we analyzed the phylogenetic relationship of the P2Y, P2Y-like, and CysLT receptors based on sequence alignment followed by evolutionary analyses. The analyses showed that the P2Y12, P2Y(13), P2Y(14), GPR87, CysLT-1, and CysLT-2 receptors formed a P2Y-related receptor subfamily with common sequence motifs in the transmembrane regions. (c) 2005 Elsevier Inc. All rights reserved.