Numerous cellular functions are influenced by mitochondrial activity, including the organelle’s ability to produce ATP, accumulate Ca²⁺ and produce reactive oxygen species. Mitochondria are distributed throughout the cytosol and, as such, these small organelles (~1-5 μm) may display particular influence over processes in their immediate neighbourhood, e.g. on nearby Ca²⁺-sensitive channels. Indeed, mitochondrial activity within sub-cellular microdomains has been implicated in the regulation of channels both on the plasma membrane and the internal calcium stores, the endoplasmic- or sarcoplasmic-reticulum (ER or SR). We have shown previously that mitochondrial uptake of Ca²⁺ promotes the activity of inositol-1,4,5-trisphosphate-sensitive Ca²⁺ release channels (IP₃R) on the SR in smooth muscle cells. This suggests a localised regulation of IP₃R by mitochondria located close to the channel, yet it has been difficult to study how mitochondria, acting in restricted regions, control cell-wide activity. We have developed a mitochondrially targeted UV-activated caged dinitrophenol. UV irradiation in vitro caused it to undergo photolysis, a product of which displayed the absorbance properties of free dinitrophenol (DNP, an uncoupler of the mitochondrial membrane potential, ΔΨ_m, from ATP synthesis). Smooth muscle cells freshly isolated from guinea-pig colon were loaded with mitochondrially targeted UV-activated caged dinitrophenol (200 nM) plus the ΔΨ_m-sensitive dye tetramethylrhodamine ethyl ester (TMRE, 10 nM) for 30 min. High-speed epifluorescent imaging detected localised decreases in TMRE fluorescence specifically in regions (as small as 5 μm diameter) exposed to UV laser light for periods <1s. The regions of decreased TMRE fluorescence only spread slightly over the course of an experiment (~20 min), i.e. the uncoupling remained localised to the site of photolysis. No changes in TMRE fluorescence were observed to UV light in the absence of the caged uncoupler . These results indicate that, in freshly isolated colonic smooth muscle cells, mitochondria are individual, electrically-independent units that do not move throughout the cell. The caged dinitrophenol has the benefits of being membrane permeable (hence alleviating the requirement for micro-injection or patch-clamping) and targeted to mitochondria, thus refining the capability to rapidly depolarise ΔΨ_m in very small sub-cellular regions and determine the role of those mitochondria in cellular signalling processes.