There are two types of mechanosensitive hair cell in the mammalian cochlea: outer hair cells (OHCs), implicated in mechanical tuning of the sound responsiveness of the basilar membrane, and inner hair cells (IHCs), the primary sensory end cells of the auditory nerve fibres. The two cells types are distinguishable morphologically, by the organization of their stereocilial bundle and by their position within the organ of Corti. Both types of hair cell are innervated by afferent dendrites through ribbon synapses, with OHC and IHC pathways projecting to different brainstem targets. In the mouse cochlea, each IHC contacts between 10 and 20 afferent dendrites, depending on cochlear position, and it has been a long-standing problem to identify the significance of this signal pathway divergence. In order to study possible functional differences between individual ribbon synapses in IHCs, we have developed imaging techniques using two photon laser scanning confocal microscopy to look into the organ of Corti in the isolated temporal bone, (Griesinger et al, 2005). Using FM1-43 as marker of intracellular vesicle and membrane trafficking from the apex to the base of mouse IHCs, we found it possible to identify regions of intense fluorescence, each about 200-500 nm in diameter, localized at the base of each IHC. Such regions are candidate sites of vesicle aggregation around the ribbon. Combined with whole cell patch clamp recording, individual calcium entry sites around the cell’s basal pole could also be detected using conventional calcium indicators (e.g OGB-5N) added through the patch pipette. On depolarization of an IHC from -70 to -10 mV, calcium increased in tightly controlled regions (‘hotspots’) near the basal pole of the IHC. Up to five spots could be imaged simultaneously in any one plane. The kinetics showed a rapid rise to a peak and decay with a time constant of ca. 200 ms. The positions of such calcium signals were compatible with colocalisation of calcium entry and the ribbon structures. Individual responses varied in amplitude from one side of the IHC to the other. In mean, 34% larger amplitude responses were found on the modiolar side as compared to the pillar side of the cell. The mechanism underlying this difference is not clear, but is likely to correlate with the distinction between the sites of origin of high and low threshold auditory nerve fibres terminating on the IHCs found in other species (Liberman, 1982). This heterogeneity of synaptic properties has been implicated explaining how a full range of sound intensities can be encoded by the cochlea.