Stimulation of receptors on the surface of animal cells often evokes cellular responses by raising intracellular Ca(2+) concentration. The rise in cytoplasmic Ca(2+) drives a plethora of processes, including neurotransmitter release, muscle contraction, and cell growth and proliferation. Mitochondria help shape intracellular Ca(2+) signals through their ability to rapidly take up significant amounts of Ca(2+) from the cytosol via the uniporter, a Ca(2+)-selective ion channel in the inner mitochondrial membrane. The uniporter is subject to inactivation, whereby a sustained cytoplasmic Ca(2+) rise prevents further Ca(2+) uptake. In spite of its importance in intracellular Ca(2+) signaling, little is known about the mechanism underlying uniporter inactivation. Here, we report that maneuvers that promote matrix alkalinisation significantly reduce inactivation whereas acidification exacerbates it. We further show that the F(1)F(0)-ATP synthase complex is an important source of protons for inactivation of the uniporter. These findings identify a novel molecular mechanism that regulates the activity of this ubiquitous intracellular Ca(2+) channel, with implications for intracellular Ca(2+) signaling and aerobic ATP production.