Recent evidence, from a variety of cell types, suggests that mitochondria play an important role in shaping the change in intracellular calcium concentration ([Ca2+]i) that occurs during physiological stimulation. In the present study, using a range of inhibitors of mitochondrial Ca2+ uptake, we have examined the contribution of mitochondria to Ca2+ removal from the cytosol of smooth muscle cells following stimulation. In voltage-clamped single smooth muscle cells, we found that following a 8-s train depolarizing pulses, the rate of Ca2+ extrusion from the cytosol was reduced by more than 50% by inhibitors of cytochrome oxidase or exposure of cells to the protonophore carbonyl cyanide P-trifluoromethoxy-phenylhydrazone. Using the potential-sensitive indicator-tetramethyl rhodamine ethyl ester, we confirmed that the effect of these agents was associated with depolarization of the mitochondrial membrane. Since, the primary function of the mitochondria is to provide the cell's ATP, it could be argued that it is the ATP supply to the ion pumps which is limiting the rate of Ca2+ removal. However, experiments carried out with the mitochondrial Ca2+ uniporter inhibitor ruthenium red produced similar results, while the ATP synthetase inhibitor oligomycin had no effect, suggesting that the effect was not due to ATP insufficiency. These results establish that mitochondria in smooth muscle cells play a significant role in removing Ca2+ from the cytosol following stimulation. The uptake of Ca2+ into mitochondria is proposed to stimulate mitochondrial ATP production, thereby providing a means for matching increased energy demand, following the cell's rise in [Ca2+]i, with increased cellular ATP production.