Abstract Body: Mitochondria use the proton motive force created by the activity of the electron transport chain to produce ATP. Thus, ionic movement across the inner membrane is restricted and strictly controlled to avoid dissipating the mitochondrial membrane potential. Unlike electroneutral mitochondrial transporters, electrogenic transporters are scarce and few such transporters or ion channels have been identified. Here, we report the presence of a new mitochondrial current. While recording inner-membrane currents in mitoplasts (mitochondria devoid of the outer membrane) in MCU knockout (Mcu^-/-) hearts, we observed an outward rectifying cationic current activated with voltage-ramps under symmetric divalent-free pipette and bath solutions. We saw this current in mitochondria from various organs including liver, brain, spleen, and the heart, and in cultured cell lines (HEK293T and Hap1). This ubiquitous current, named here LUNA (Large, Ubiquitous, Non-selective, Amiloride-sensitive channel), is a non-selective cation current (K⁺ > Na⁺ > Ca²⁺ ≈ Mg²⁺ ≈ NMDG⁺) active at depolarized membrane potentials (≥0 mV), a condition that normally leads to the activation of proapoptotic mechanisms. We observed that the basal activity of LUNA currents in wild type mice hearts augmented upon chelation of external Ca²⁺ and Mg²⁺. Moreover, the activity of LUNA was enhanced approximately three-fold in both the cardiac-specific knockout of the nuclear-encoded mitochondrial transcription factor Tfam (Tfam cKO), a model for mitochondrial cardiomyopathies, and in mice with cardiac pressure overload due to transverse aortic constriction compared to the respective controls. Importantly, we were able to observe LUNA current in human end-stage heart failure patients (HFrEF), with near nanoampere amplitudes, making LUNA the largest-ever current directly recorded from mitochondria. Taken together, our results indicate that LUNA is expressed in mammals, and that LUNA amplitude increases during a variety of forms of human or murine cardiac injury.