The inner hair cell (IHC) is the primary sensory cell of the mammalian cochlea, each cell forming multiple synaptic contacts with the auditory nerve. Their ribbon synapses are characterised by dense bodies surrounded by numerous vesicles and capable of multivesicular release precisely linked to depolarization induced by the sound stimulus (Goutman & Glowatzki, 2007). The mechanism for the control of release is incompletely understood. To this end we have been using multiphoton imaging to identify the calcium entry sites near the ribbon when the cell is depolarized under whole cell voltage clamp. Cochlear IHCs were imaged and recorded using an in situ preparation of the CaJ mouse inner ear allowing adult tissue to be studied whilst preserving cochlear geometry. Animals aged P28 and above were used. The temporal bone was removed after killing the animals by cervical dislocation and stuck to the base of a chamber containing artificial perilymph (in mM, NaCl 135, KCl 5.4, MgCl2 1, CaCl2 1.3, Na2HPO4 0.7, hepes 10, to pH 7.3). Access to the cells was possible through a small opening in the apical turn which exposed the 5-15 kHz region of the cochlear spiral. The ribbon constituent peptide CtBP2 was subsequently identified by immunohistochemistry. In this region we found 6 -20 ribbons per IHC (mean 12.2 ± 2.9 S.D., n = 83 IHCs) using reconstructed z-stacks to identify the ribbon distribution. IHCs were recorded with pipettes containing K+ channel blockers to reduce the large outward currents. Pipettes contained (in mM): CsMeSO3 130, TEACl 13, EGTA 1, Mg2ATP 3, hepes 5, to pH 7.3). OGB-5N (200 μM), a low affinity fluorescent indicator, was included in order to measure intracellular calcium. Inward calcium currents could be recorded on depolarization from -60 to 0mV. In many cases, simultaneous imaging the of the basal pole of the IHC with the multiphoton upright microscope, exciting the dye at 935 nm, revealed large increases in the baseline fluorescence. The largest increases occurred at highly localised regions (‘hotspots’). Variability in the size of individual responses was partly ascribed to different amplitude calcium signals arising from presumed ribbon sites at opposite sides of the IHC (Culley & Ashmore, 2010). The voltage dependence of the peak calcium signal at different hotspots was indistinguishable, suggesting the underlying calcium channel type to be identical. Calcium signals also increased with the duration of depolarization and spreading along the IHC axis by about 3 μm for prolonged depolarizations (500 ms). In some cells, a 10ms depolarization generated a calcium signal with ΔF/F=0.27, rising to 3.2 for 200 ms steps. The results are compatible with a calcium entry highly localised at the ribbon synapse, and strongly buffered around the site of entry into the IHC.