Lack of permeable inhibitors of the mitochondrial Ca²⁺ uniproter has precluded in the past investigating directly the physiological implications of mitochondrial Ca²⁺ uptake in the global cell Ca²⁺ signalling. We show here that the thiourea derivative KB-R7943, previously used as inhibitor of the plasma membrane Na⁺/ Ca²⁺ exchanger, is a potent inhibitor of the mitochondrial Ca²⁺ uniporter (MCU) active both in intact and permeabilized cells (Ki 5.5 ± 1.3 mM, mean ± S.D.). KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na⁺/ Ca²⁺ exchanger. Given that non-excitable cells lack most of the alternative targets of this inhibitor, the availability of a permeable inhibitor of MCU allows studying the effect of selective MCU inhibition on Ca²⁺ release and cytosolic Ca²⁺-oscillations. In intact cells, 10mM KB-R7943 reduced by about 80% the mitochondrial [Ca²⁺] peak induced by histamine. KB-R7943 inhibited the fast phase of Ca²⁺-release from the endoplasmic reticulum, but enhanced the subsequent slow one. Consistently, the peak of cytosolic [Ca²⁺] induced by histamine was reduced but the subsequent steady-state phase was increased. KB-R7943 did not modify histamine-induced Ca²⁺-release in cells loaded with a Ca²⁺ chelator, suggesting that its inhibitory effect on Ca ²⁺-release is mediated by an increase in feedback Ca²⁺ inhibition of inositol 1,4,5-trisphosphate receptors following MCU block. When tested on histamine-induced cytosolic [Ca²⁺] oscillations, KB-R7943 reversibly blocked oscillations in the same range of concentrations required to inhibit MCU, providing new evidence for the role of mitochondria modulating Ca²⁺ release and regenerative Ca²⁺ oscillations.