Changes in the cytosolic calcium concentration ([Ca²⁺]_c) control a diverse array of cellular functions. [Ca²⁺]_c can be elevated either by influx of extracellular Ca²⁺,for example following activation of voltage-gated Ca²⁺ channels, or by release of Ca²⁺ from intracellular stores. The major Ca²⁺ store in smooth muscle cells is the sarcoplasmic reticulum, from which Ca²⁺ release occurs via either inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) or ryanodine receptors (RyR). Although thought of primarily as the site of ATP generation, mitochondria also take up Ca²⁺,modulating Ca²⁺ influx or release via IP₃R or RyR. Indeed recent evidence has suggested that some mitochondria may be closely apposed to IP₃R, such that mitochondrial Ca²⁺ handling may have very localised effects on nearby IP₃R. Hence the contribution of mitochondria to IP-mediated Ca²⁺ signals was examined in single smooth muscle cells, freshly dissociated from guinea pig colon (humanely killed by stunning and exsanguination) and patch-clamped in the whole-cell configuration.The IP₃-generating agonist carbachol (CCh) or IP₃ release by UV flash photolysis of the caged compound introduced via the patch pipette evoked large [Ca²⁺]_c transients. Depolarisation of the mitochondrial membrane potential (∆ψ _m) with CCCP plus oligomycin, or rotenone plus oligomycin inhibited IP₃-induced [Ca²⁺]_c transients (peak declined to 47.1 ± 8.86 % of control values in CCCP plus oligomycin, or 49.3 ± 9.86 % in rotenone plus oligomycin, n=11 for each; p<0.01 by unpaired Students t-test, data expressed as mean ± SEM) or CCh-induced [Ca²⁺]_c transients (peak declined to 8.32 ± 6.45 % in CCCP plus oligomycin, or 61.78 ± 9.27 % in rotenone plus oligomycin, n=3 for each; p<0.01). This indicates that mitochondrial Ca²⁺ uptake did occur following IP₃– or CCh-induced [Ca²⁺]_c elevation. Simultaneous imaging of [Ca²⁺]_c and ∆ψ in single myocytes co-loaded with the Ca²⁺ indicator fluo-4 AM and the membrane potential sensitive dye TMRE indicated that there was no detectable alteration in ∆ψ during IP₃– or CCh-induced [Ca²⁺]_c elevation. Inhibition of the mitochondrial Ca²⁺ efflux pathway, the mitochondrial Na⁺/Ca²⁺ exchanger, with CGP-37157 increased mitochondrial Ca²⁺ content and both the time to maximum [Ca²⁺]_c and the time for [Ca²⁺]_c to fall back to starting values following IP₃ release. Subsequently there was also an IP₃-release dependant elevation of the baseline [Ca²⁺]_c and a decline in the maximum IP₃-evoked [Ca²⁺]_c.These results suggest that mitochondrial Ca²⁺ uptake modulates the IP₃-mediated Ca²⁺ signal. Mitochondrial Ca²⁺ uptake occurs without significant mitochondrial depolarisation and Ca²⁺ export via the mitochondrial Na⁺/Ca²⁺ exchanger is essential for maintenance of mitochondrial regulation of IP₃-mediated Ca²⁺ signals.