AbstractCa²⁺ influx triggers the release of synaptic vesicles at the presynaptic active zone (AZ). A quantitative characterization of presynaptic Ca²⁺ signaling is critical for understanding synaptic transmission. However, this has remained challenging to establish at the required resolution. Here, we employ confocal and stimulated emission depletion (STED) microscopy to quantify the number (20–330) and arrangement (mostly linear 70 nm × 100–600 nm clusters) of Ca²⁺ channels at AZs of mouse cochlear inner hair cells (IHCs). Establishing STED Ca²⁺ imaging, we analyze presynaptic Ca²⁺ signals at the nanometer scale and find confined elongated Ca²⁺ domains at normal IHC AZs, whereas Ca²⁺ domains are spatially spread out at the AZs of bassoon-deficient IHCs. Performing 2D-STED fluorescence lifetime analysis, we arrive at estimates of the Ca²⁺ concentrations at stimulated IHC AZs of on average 25 µM. We propose that IHCs form bassoon-dependent presynaptic Ca²⁺-channel clusters of similar density but scalable length, thereby varying the number of Ca²⁺ channels amongst individual AZs.