Each inner hair cell (IHC) of the mammalian cochlea contains a cluster of 10-20 ribbon synapses specialised for rapid release of neurotransmitter. Each synapse controls the firing in a single afferent nerve, and paired recording from pre- and postsynaptic sites show that, following an initial high rate of release, each ribbon synapse is able to maintain a sustained level of vesicle fusion (Goutman & Glowatzki, 2007). In vivo recording of auditory nerve fibres show that not all fibres have comparable thresholds to sound (e.g Taberner & Liberman, 2005) suggesting local differences between IHC synapses. To investigate this system we have used simultaneous whole cell recording and calcium imaging in IHCs in the organ of Corti of adult (P21-P60) mice. The temporal bone was removed after killing the animals by cervical dislocation and was then stuck to the base of the chamber. A small opening made in the apical cochlea to allow access for patch recording and imaging of the basal synaptic pole of the cell using 2 photon laser scanning confocal microscopy equipped with a 63x NA 1.0 WI objective. The pipette contained (in mM) CsCl, 140; EGTA, 1; MgCl2, 2; OGB1 or OGB5N, 0.25; and hepes, 10; pH 7.3. The caesium blocked all but 1-2 nA of the large outward K+ currents at 0mV revealing, at room temperature, a small (60-80 pA ) inward Ca2+ current at -30 mV. Using 935 nm excitation for the calcium indicator, with maximum frame rates of 70 Hz, fluorescence increased at highly localised sites. 100 ms IHC depolarizations from -70 to -20 mV elicited a rapid rise of a signal and decay over approximately 200 ms. In some cases the signal appeared to rise to a local sustained peak, explained by the proximity of the entry site to the PSF of the objective. The typical mean diameter of the spots was 0.65 μm, suggesting a Ca2+ entry area not more than 0.5 μm in diameter. With imaging planes close to the IHC basal pole, up to six localised ‘hotspots’ could be detected in one section, consistent with ribbon counts determined by immunohistochemisty (Meyer et al, 2009). Although there was variation between cells, the peak amplitude of the responses differed between distinct regions of a single IHC. Normalised with respect to background, responses recorded from the modiolar side of the IHC base (n=29 ‘hotspots’) were 1.34 times larger than from the abneural side (n=17 ‘hotspots’). The results support the hypothesis that variation in organised calcium control between ribbon synapses within a single IHC may determine the differential sensitivity of individual auditory nerve fibres.