Mitochondrial Ca²⁺ (mCa²⁺) handling is an important regulator of liver cell function, controlling events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca²⁺ enters mitochondria through the ruthenium red-sensitive mCa²⁺ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca²⁺ channels in the cell membrane requires phospholipase C (PLC). This activation commonly occurs through phosphitidylinositol-4,5-biphosphate hydrolysis and the production of the second messengers inositol triphosphate (IP₃) and diacylglycerol. Phosphitidylinositol-4,5-biphosphate was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa²⁺ uptake through the uniporter. Western blots with anti-PLC antibodies demonstrated the presence of PLC-1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa²⁺ uptake when whole mitochondria were incubated at 37°C with ⁴⁵Ca²⁺. Increasing extra-mitochondrial [Ca²⁺] significantly stimulated mCa²⁺ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mitochondrial Ca²⁺ uptake, while U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa²⁺ uptake in any experimental condition. Membrane-permeable IP₃-receptor antagonists 2-Aminoethoxydiphenylborate and Xestospongin C also inhibited mCa²⁺ uptake. Although extra-mitochondrial IP₃ had no effect on mCa²⁺ uptake, membrane-permeable diacylglycerol analogs 1-oleoyl-2-acetyl-sn-glycerol and diacylglycerol-lactone, which inhibit PLC activity, dose-dependently inhibited mCa²⁺ uptake. These data indicate that PLC-1 exists in liver mitochondria and is involved in regulating mCa²⁺ uptake through the uniporter.