Mitochondrial dysfunction has been implicated in age related hearing loss and in the response to both noise damage and ototoxins. Documentation of mitochondrial function in the inner ear however is scarce and little is known about their role in cochlear physiology and pathophysiology. Recent advances in techniques available for the study of mitochondria have furthered our understanding of their biochemical and bioenergetic functions in single cell systems. In addition to ATP production, mitochondria participate in numerous other processes within the cell; including Ca²⁺ homeostasis, regulation of intracellular redox potential and cell death. The cochlea is a complex, multicellular organ known to have a high metabolic demand. It exhibits a tonotopic gradient such that high frequency sounds are encoded at the basal end and low frequency sounds at the apex. There is also a base to apex gradient in the sensitivity of sensory hair cells to noise and ototoxins. Explant cultures were prepared from postnatal day 3 or 4 rat cochleae and used after 1 day in vitro. In addition, cochlear slices were prepared from postnatal cochleae and used within a few hours. The mitochondrial transmembrane potential (ψ_mt) is central to oxidative phosphorylation. We assessed ψ_mt using the fluorescent lipophilic cation tetrametyl rhodamine methyl ester (TMRM). Analysis of mitochondrial TMRM fluorescence suggests a difference in ψ_mt in inner hair cells from the base of the cochlea compared to the apex. To further investigate differences in base-to-apex mitochondrial physiology, we used the endogenous autofluorescence of NAD(P)H and FAD. Measurement of NAD(P)H and FAD autofluorescence provides an assessment of mitochondrial metabolism. The spectral properties of the two signals enable them to be imaged simultaneously using excitation with 351 nm and 488 nm laser lines. To calibrate the signals, 3 mM NaCN was applied to inhibit mitochondrial respiration and induce a state of maximal reduction. This was followed by 5 µM FCCP, an uncoupler that collapses ψmt and establishes a state of maximal oxidation. Calibration of the signals in this way provides an estimation of cell redox state. We are presently continuing this work in order to confirm the presence of base-to-apex differences in mitochondrial function in subtypes of cochlear cells.