The mammalian cochlea contains a population of sensory cells, inner hair cells (IHCs), which act as the input cells to the auditory system. The IHCs signal to the auditory system through ribbon synapses, each cell having multiple release sites onto single auditory neurites. It is generally thought that the sharp neural tuning seen in single fibres in hearing animals reflects the selectivity of individual IHCs. Using a preparation which allows recording from adult hair cells, rather than immature systems more conventionally used, we have employed a fluorescent probe to investigate calcium signalling at the IHC synapse by using simultaneous multiphoton confocal imaging and cell recording. Cochlear inner and outer hair cells were visualised in the intact isolated temporal bone from mice aged between P25 and 10 months. Animals were killed by rapid cervical dislocation in accordance with UK legislation and guidelines. The apical cochlea was opened to reveal the 10-15 kHz cochlear region with the cells placed in identifiable positions and orientation. The tectorial membrane was removed to allow recording and the chamber was superfused with (in mM) Na, 140; Ca, 1.3; K, 4; Cl 147, hepes 10, phosphate 0.7 to pH 7.3 at room temperatures. Cells were recorded in the whole cell tight seal mode, the pipette including 0.5, 5 or 10 mM EGTA and 0.25 mM OGB5N. Cs was used to reduce large outward currents to less than 1.5 nA at 0 mV. These recordings showed distinct calcium entry sites, ‘hotspots’ , when the IHC was depolarized to 0 mV from -60 mV. The kinetics of the Ca rise and fall often slowed with extended recording time over 10 mins. After OGB5N loaded for 3-5 mins it was also often found that a proportion of neighbouring cells appeared to be fluorescently loaded. Up to 9 adjacent cells could be found filled with OGB5N. Recording time constants in such cases exceeded the single cell values and could be modelled on the assumption that the cells were electrically coupled with an intercellular resistance of less than 10 megohms. Calcium responses imaged in such coupled IHCs showed that the kinetics generally differed from that of the recorded cell, suggesting that the coupling did not permit the movement of large Ca buffers between cells. The present data thus indicate that adult IHCs can be coupled. The extent of the observed coupling would not significantly degrade the frequency selectivity of individual auditory nerves, at least in low frequency fibres, but may increase the signal/noise ratio in the pathways leading from the apical cochlea.