ATP is a known extracellular ligand for cell membrane purinergic receptors. Intracellular ATP can work also as a regulatory ligand via binding sites on functional proteins. We report herein the existence of P2Y(1)-like and P2Y(2)-like receptors in hepatocyte mitochondria (mP2Y(1) and mP2Y(2)), which regulate mCa(2+) uptake though the uniporter. Mitochondrial P2Y(1) activation stimulates mCa(2+) uptake; whereas, mP2Y(2) activation inhibits mCa(2+) uptake. ATP acts preferentially on mP2Y(2) receptors, while ADP and AMP-PNP stimulate both the mP2Y(1) and mP2Y(2). PPADS inhibits ADP stimulated mP2Y(1)-mediated mCa(2+) uptake. In addition, UTP, a selective P2Y(2) agonist, strongly inhibits mCa(2+) uptake. The newly discovered presence and function of these receptors is significant because it explains increased mCa(2+) uptake in the setting of low cytosolic [ATP] and, therefore, establishes a mechanism for direct feedback in which cytosolic [ATP] governs mitochondrial ATP production through regulation of mCa(2+) uptake.