Ischemia-reperfusion injury remains a major clinical problem in liver transplantation. One contributing factor is mitochondrial calcium (mCa2+) overload, which triggers apoptosis; calcium also regulates mitochondrial respiration and adenosine 5′-triphosphate (ATP) production. Recently, we reported the presence of purinergic P2Y1- and P2Y2-like receptor proteins in mitochondrial membranes. Herein, we present an evaluation of the functional characteristics of these receptors. In experiments with isolated mitochondria, specific P2Y1 and P2Y2 receptors ligands: 2-methylthio-adenosine 5′-diphosphate (2meSADP) and uridine 5′-triphosphate (UTP), respectively, were used, and mitochondrial calcium uptake was measured. 2meSADP and UTP had a maximum effect at concentrations in the range of the known P2Y1 and P2Y2 receptors. The P2Y inhibitor phosphate-6-azophenyl-2′,4′-disulfonate (PPADS) blocked the effects of both ligands. The phospholipase C (PLC) antagonist U73122 inhibited the effect of both ligands while its inactive analog U73343 had no effect. These data strongly support the hypothesis that mitochondrial Ca2+ uptake is regulated in part by adenine nucleotides via a P2Y-like receptor mechanism that involves mitochondrial PLC activation. J. Cell. Biochem. 99: 1165–1174, 2006. © 2006 Wiley-Liss, Inc.