The activity of mitochondria impacts upon many aspects of cellular physiology, including ATP production and Ca²⁺ regulation. We have shown previously that mitochondria regulate InsP₃-sensitive Ca²⁺ release channels (InsP₃R) on the intracellular Ca²⁺ store, the sarcoplasmic reticulum (SR), in freshly isolated smooth muscle cells. Depolarisation of the electrical potential across the inner mitochondrial membrane (Ψ_m) inhibited mitochondrial Ca²⁺ accumulation, which in turn inhibited Ca²⁺ release via InsP₃R. This result suggested a close interaction between mitochondria and the microdomain of Ca²⁺ released by InsP₃R; localised mitochondrial Ca²⁺ uptake may prevent a negative feedback of Ca²⁺ upon InsP₃R activity. Until now, the only method of investigating the influence that mitochondria exert over cellular processes such as Ca²⁺ signalling has been pharmacological depolarisation of all mitochondria within a cell. We have developed a caged mitochondrial inhibitor that may be released in subcellular regions of interest, by selective exposure UV light to enable depolarisation of mitochondria in subcellular regions to occur. The compound AG10 (also known as tyrphostin 8), depolarises Ψ_m by increasing the proton conductance of the inner mitochondrial membrane. AG10 was caged chemically with a 4, 5-dimethoxy-2-nitrobenyl alcohol group attached to the 6’ hydroxyl of AG10. The caged form of AG10 did not depolarise Ψ_m when introduced into the cytosol of single smooth muscle cells freshly isolated from guinea pig colon, via a whole-cell patch pipette. When AG10 was uncaged in a restricted region of the cell, Ψ_m depolarised, as determined by a loss of fluorescence of the Ψ_m-sensitive dye tetramethyl-rhodamine ethyl ester (TMRE), in this region only. This depolarisation did not spread to neighbouring mitochondria over the next 20 min but remained restricted to the region selectively illuminated by UV light. The InsP₃-generating agonist carbachol (CCh) evoked cell-wide increases in [Ca²⁺]_c, that were inhibited by localised Ψ_m depolarisation, both at the site of Ψ_m depolarisation and at distant sites where Ψ_m was unaffected. These results suggest that, in smooth muscle, the localised action of mitochondria can affect Ca²⁺ signalling, not only local to the individual mitochondria but also throughout the cell.