The collecting duct (CD) consists of two different cell populations, principal cells (PCs) and intercalated cells (ICs), which have distinct solute transport functions. Given that transport and metabolism are closely coupled in the nephron, we hypothesise that there are intrinsic differences in mitochondrial function between PCs and ICs; this has not previously been investigated due to a lack of appropriate technology. It has been demonstrated that mitochondrial function can be measured in situ in intact rat kidney slices using confocal and multiphoton microscopy (1); we applied this approach to investigate our hypothesis. Adult male Sprague-Dawley rats were killed by cervical dislocation (in accordance with the Animals (Scientific Procedures) Act 1986). Kidneys were removed immediately, decapsulated and placed in oxygenated ice-cold physiological saline solution. Serial 200 µm-thick coronal kidney slices were cut using a vibrotome slicer (2) and fluorescent dyes were loaded using a re-circulating perfusion system gassed with 95% O2 5% CO2. Slices were imaged with a Zeiss LSM 510 NLO axiovert microscope, coupled to a tunable Coherent Chameleon laser and remained viable for up to 4h, confirmed by calcein-AM dye uptake. The two cell types have distinct morphological differences, which facilitated their individual identification. Antibodies to label PCs and mitochondria (aquaporin 2 and ATP5β respectively) confirmed differences in cell morphology and revealed apical distribution of mitochondria in ICs. Marked heterogeneity was observed in mitochondrial signals along the CD. Mitochondrial membrane potential, as measured using the lipophilic cation tetramethylrodamine methyl ester (TMRM), was higher in PCs (mean TMRM signal: 34.45 A.U. ±1.43) than ICs (16.82±0.28, n=3, p=0.009). The differential mitochondrial potential observed was not altered by the addition of dye efflux inhibitor verapamil or the ATP-synthase inhibitor oligomycin indicating that the signal seen was not artefactual. Autofluorescence signals emitted by NADH and FAD2+ (respective substrates for complexes I and II in the respiratory chain) were both higher in ICs than in PCs (p=0.03 and p=0.056 for NADH and FAD2+ respectively). Reactive oxygen species production was measured using fluorescent dihydroethidium (HEt) and a higher signal detected in ICs. Glutathione, an important intracellular antioxidant, was measured using monochlorobimane (MCB), which forms a fluorescent adduct with glutathione. Once MCB signal intensities had reached a steady state (40 minutes), glutathione levels were higher in PCs (mean MCB fluorescence 170.84±10.53 A.U.) than in ICs (43.73±10.96) (n=3, p=0.006). In summary, fluorescence imaging reveals intrinsic differences in mitochondrial function between PCs and ICs, which may be important in understanding physiology and patho-physiology in this nephron segment.