For normal cochlear function, outer hair cells (OHCs) require a precise control of intracellular Ca2+ levels. In the absence of regulatory elements such as proteinaceous buffers or extrusion pumps, OHCs degenerate, leading to profound hearing impairment. Influx of Ca2+ occurs both at the stereocilia tips and the basolateral membrane. In this latter compartment, two different origins for Ca2+ influx have been poorly explored: voltage-gated Ca2+ channels (VGCC) at synapses with type II afferent neurons, and α9α10 cholinergic nicotinic receptors at synapses with medio-olivochlear complex (MOC) neurons. Using functional imaging in mouse OHCs, we dissected Ca2+ influx individually through each of these sources, either by applying step depolarizations to activate VGCC, or stimulating MOC axons. Ca2+ ions originated in MOC synapses, but not by VGCC activation, was confined by Ca2+-ATPases most likely present in nearby synaptic cisterns. Although Ca2+ currents in OHCs are small, VGCC Ca2+ signals were comparable i...