In the cochlea, cell damage triggers intercellular Ca2+ waves that propagate through the glial-like supporting cells that surround receptor hair cells. These Ca2+ waves are thought to convey information about sensory hair cell-damage to the surrounding supporting cells within the cochlear epithelium. Mitochondria are key regulators of cytoplasmic Ca2+ concentration ([Ca2+](cyt)), and yet little is known about their role during the propagation of such intercellular Ca2+ signalling. Using neonatal rat cochlear explants and fluorescence imaging techniques, we explore how mitochondria modulate supporting cell [Ca2+](cyt) signals that are triggered by ATP or by hair cell damage. ATP application (0.1-50 microM) caused a dose dependent increase in [Ca2+](cyt) which was accompanied by an increase in mitochondrial calcium. Blocking mitochondrial Ca2+ uptake by dissipating the mitochondrial membrane potential using CCCP and oligomycin or using Ru360, an inhibitor of the mitochondrial Ca2+ uniporter, enhanced the peak amplitude and duration of ATP-induced [Ca2+](cyt) transients. In the presence of Ru360, the mean propagation velocity, amplitude and extent of spread of damage-induced intercellular Ca2+ waves was significantly increased. Thus, mitochondria function as spatial Ca2+ buffers during agonist-evoked [Ca2+](cyt) signalling in cochlear supporting cells and play a significant role in regulating the spatio-temporal properties of intercellular Ca2+ waves.